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Previously, we described a defective Autographa californica nuclear polyhedrosis virus (AcMNPV) which must contain cis-acting elements required for DNA synthesis, such as the origin(s) of replication (ori). Defective genomes of AcMNPV generated after serial undiluted passage were analyzed further. Three small separated regions were retained in DNA of defective AcMNPV and accumulated in extracellular defective interfering viruses as well as in intracellular DNA after 40 passages. Two of these regions have now been identified as containing putative ori. They are located on the HindIII-B fragment between map units (m.u.) 50.1 and 53.2 and on the HindIII-Q fragment between m.u. 87.2 and 88.9 of the physical map of AcMNPV DNA, respectively. Transfection of Spodoptera frugiperda cells with plasmids containing these sequences followed by superinfection with intact helper AcMNPV resulted in amplification of these plasmids, as demonstrated by the Dpnl sensitivity assay. The replicating activity of HindIII-Q is putatively located within the 1000-bp region containing a highly repetitive DNA (hr5), which is also ascribed to enhance delayed-early gene expression. In order to demonstrate replicating activity of test plasmids, it appeared essential to transfect the cells well before superinfection with helper virus.