To examine the status of cell cycle-inhibitory genes in human prostate carcinoma, we investigated alterations of RE (retinoblastoma), p16/CDKN2 and p15(INK4B) genes in 32 adenocarcinomas with immunohistochemistry. PCR-single-strand conformation polymorphism (SSCP) was used to examine all 27 exons of the RE gene, exons 1 to 3 of the p16/CDKN2 gene and exons 1 and 2 of the p15(INK4B) gene for mutations. Loss of heterozygosity (LOH) for the RE gene was probed by restriction fragment length polymorphism (RFLP) analysis. In addition, coordinate samples were subjected to immunohistochemical studies for reactivity to RE and p16 protein. The RE gene alterations were detected in 5 of the 32 tumors (16%); of these, only one mutation, a missense substitution, occurred within an exon. The remaining four single base insertions or deletions were found within introns of the RE gene and no mutational event was detected in its promoter region. LOH involving intron 17 of RB was detected in three cases of 10 informative tumors (30%). Intragenic mutations were also present in 3 of the 32 tumors in the p16/CDKN2 gene. In contrast, no mutational events were found in the p15(INK4B) gene in the tumors. Only one tumor had both a p16/CDKN2 mutation and LOH of the RE gene. Expression of pRB was absent or reduced in 16 cancers, while p16 expression was present in all cases to varying degrees. The results suggest that p16/CDKN2 gene mutations occur rarely and intragenic mutation, but not LOH,of the RE gene is not required in prostatic tumorigenesis.