OBJECTIVE Cell-cycle inhibitor and tumor-suppressor gene p16/MTS1 was found to be altered in a variety of human tumors. To directly investigate genetic alterations and expression of p16/MTS1 and p15/MTS2, this study surveyed pancreatic tumors. METHODS Cell-cycle inhibitors were analyzed for genetic alterations and expression by polymerase chain reaction, DNA sequencing, reverse-transcription polymerase chain reaction, and Western blotting. RESULTS The analysis of pancreatic adenocarcinoma (19 cell lines and 3 xenografts) for p16/MTS1 and p15/MTS2 revealed homozygous deletions in 10 of 22 cases (46%) (7 cell lines and 3 xenografts) involving both genes. We show in these 7 cell lines as well as in 3 additional cases (10 of 19[53%]) loss of p16/MTS1 transcripts and in 2 further cases (12 of 19 [63%]) mutations leading to the loss of p16 protein. The frequency of mutations in p16/MTS1 was 56% (5 of 9). In contrast to p16/MTS1, p15/MTS2 transcripts were obtained in all cases exhibiting the p15/MTS2 gene (54%). Loss of expression was not observed for p27 and p18. CONCLUSIONS These results support that loss of p16 function could be involved in pancreatic cancer and may explain at least in part the aggressive behavior of this tumor type.