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The p16^INK4A gene encodes 2 cell cycle regulator proteins, p16 and p14^ARF, by alternative splicing. This genetic locus also contains another cell cycle regulator gene, p15^INK4B, which encodes p15. The inactivation of the p16 protein has been demonstrated in some hepatocellular carcinomas (HCCs); however, the inactivation of the other 2 cell regulator proteins and their inactivation patterns are not well characterized. To characterize the role of the above 3 cell cycle regulator proteins in HCCs, the authors examined the genomic status of the p16^INK4A and p15^INK4B genes and their RNA products in 20 HCC tissues and 7 human HCC cell lines. Homozygous deletions in each exon of p16^INK4A and p15^INK4B were evaluated by comparative multiplex polymerase chain reaction (PCR), and the methylation status of the p16^INK4A and p15^INK4B promoter region was analyzed by methylation specific PCR. Homozygous deletions were found in 6 of 20 HCCs (30%) and 2 of 7 HCC cell lines (29%). In 20 HCCs, the frequency of homozygous deletions was 20% in exon 1 of p15^INK4B, 20% in exon 2 of p15^INK4B, 10% in exon 1β of p16^INK4A, 25% in exon 1α of p16^INK4A, 15% in exon 2 of p16^INK4A, and 15% in exon 3 of p16^INK4A. The authors found hypermethylation of the p16^INK4A promoter region in 7 HCCs (35%) and 3 HCC cell lines (43%). The overall frequency of p16 alterations in HCCs, including hypermethylation and homozygous deletions, was 60% (12 of 20 cases). According to reverse transcriptase–PCR analysis, the absence of RNA expression was most frequent in p16 (11 of 20 cases, 55%) and less frequent in p15 (7 of 20 cases, 35%) and p14^ARF (5 of 20 cases, 25%). Among the 3 cell cycle regulator proteins encoded at the 9p21 genetic locus, inactivation of p16 is the most frequent event in HCCs in which promoter hypermethylation and homozygous deletions are the common mechanisms. Cancer 2000;89:60–8. © 2000 American Cancer Society.