The molecular pathology of human pancreatic cancer is poorly understood, particularly with regard to the role of known tumor-suppressor genes. We have examined the expression of the p53 and Rb-1 tumor-suppressor genes in seven human pancreatic carcinoma cell lines and 10 primary pancreatic carcinomas. Examination of the Rb-1 gene by Northern hybridization and immunoprecipitation analyses revealed the absence of Rb-1 protein expression in two cell lines. Moreover, regions of absent nuclear staining in two primary pancreatic carcinomas were detected by immunohistochemical analysis. Investigation of p53 by Southern, Northern, immunohistochemical and immunoprecipitation analyses revealed multiple abnormalities, including gross rearrangements in two cell lines, the absence of detectable p53 transcript in two cell lines and a truncated transcript in one line. Six cell lines overexpressed p53 protein, while one line revealed the absence of p53 product by immunohistochemical and immunoprecipitation analyses. Sequence analysis of exons 5-8 of the p53 gene confirmed these analyses, revealing missense mutations in all seven cell lines in codons 181, 220, 248, 249, 265, 272 and 273. Of 10 mutations identified, nine were transitions and 50% were in codon 273. Immunohistochemical analyses of frozen primary pancreatic carcinomas revealed positive nuclear staining for p53 in 40% of cases. Mutations were identified in codons 238 and 286 and in intron 9 in several representative specimens. Alterations in the p53 and Rb-1 genes may be important features in the development of human pancreatic cancer.