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Mitochondria arose from the endosymbiosis of a bacterium related to contemporary members of Alphaproteobacteria (2). Despite a probable monophylogenetic origin and the conservation of most of the biological roles of mitochondria, mitochondrial (mt) genome organization and expression are extraordinarily diverse among eucaryotes. As exemplified in animals and higher plants, mt genome size can vary from 16 kbp to several hundreds of kilobase pairs, respectively. This large increase in plant mt genome size is not correlated with increasing coding capacity but is rather explained by the presence of large intergenic regions, which are virtually absent from animal mt genomes. Plant mt genomes also contain insertions of nuclear and plastid sequences as well as foreign DNA from viral and unknown origins. As a consequence of the diversity in genome organization, mechanisms controlling mt gene expression also differ markedly between organisms. Transcription of the human mt genome is a relatively straightforward process, as both strands are transcribed from single promoters lying in the noncoding regulatory region. By contrast, numerous promoters are scattered along both strands of plant mt genomes. Moreover, even multiple promoters for a single gene are a common feature in plant mitochondria (11, 14). The recent analysis of these promoter sequences in Arabidopsis thaliana also revealed a somehow relaxed promoter specificity, as multiple sequences are able to initiate transcription (11). In addition, there is so far no evidence for a transcription termination mechanism in plant mitochondria. For instance, large regions downstream of rrn genes are expressed, although they do not give rise to stable transcripts (4). The emerging picture is that transcription in plant mitochondria is a relaxed process which, in general, exhibits little control or modulation. Rather, posttranscriptional events such as processing and control of RNA stability account for proper control of gene expression in plant mitochondria (4, 7, 13). Exoribonucleases are major players in RNA 3′ processing and degradation processes. We have recently characterized an A. thaliana nuclear gene (At5g14580) that encodes a mitochondrial exoribonuclease belonging to the polynucleotide phosphorylase (PNPase) family (17). The mt PNPase is essential for viability in Arabidopsis, in contrast to the chloroplast PNPase-like protein encoded by the At3g03710 gene. Down-regulation of the mt PNPase results in the accumulation of unprocessed RNAs, such as atp9 and orfB mRNA and 18S rRNA precursors (16, 17). Moreover, RNA species that are quickly turned over in wild-type (WT) plants accumulate in the absence of PNPase. Such RNAs include, for instance, 18S rRNA degradation intermediates and the leader of the 18S rRNA, which is removed by an endonuclease from the primary transcript. These initial studies revealed that PNPase is essential for several aspects of mt RNA metabolism, including 3′ processing and degradation processes. Interestingly, all RNA substrates of PNPase that we have investigated so far are polyadenylated. Although mature RNAs are not constitutively polyadenylated in plant mitochondria, poly(A) tails trigger rapid exonucleolytic degradation by PNPase, similar to the situation reported first for Escherichia coli and later for chloroplasts (1, 3). To determine new substrates, and thus novel biological roles, of PNPase, we cloned about 300 polyadenylated mt RNAs from plants down-regulated for PNPase (PNP− plants). These sequences were considered degradation tags, as they allowed the identification of different classes of transcripts requiring PNPase for their degradation as judged by their accumulation in PNP− versus WT plants. Our results indicate that maturation by-products such as rRNA leaders or tRNA intergenic sequences are generally degraded by PNPase. In addition, we show that a major role for PNPase is the degradation of transcripts that are, in some cases, expressed to surprisingly high levels from regions lacking known genes. Some of these RNAs, which include transcripts of chimeric open reading frames (ORFs) created by mt DNA recombination events or antisense (AS) RNA transcribed from the opposite DNA strand of a known gene, could have a deleterious effect on mitochondrial function. These data show that the lack of tight transcriptional control in Arabidopsis mitochondria is counterbalanced by polyadenylation-mediated decay by PNPase.