To identify whether alterations of the p16 tumor suppressor gene are a common event in localized prostate cancer, we examined the frequency of p16 gene mutations in 30 primary tumors. Only two tumors demonstrated altered single-strand conformation polymorphism patterns for exon 2 of p16. In both cases, sequencing revealed a missense at codon 148, a GβA transition that resulted in the replacement of the alanine by threonine. Polymerase chain reaction-single-strand conformation polymorphism analysis of matched blood samples revealed the same abnormal band shifts as the tumor samples, suggesting that these base changes are polymorphic. In addition, transcriptional inactivation by means of CpG island methylation has also been reported as a possible means of p16 gene inactivation. To address this point, we determined the pattern of DNA methylation at the SmaI site for 21 of 30 samples for which DNA was available. Only one sample had an altered methylation pattern at the SmaI site downstream of exon 1 of the p16 gene, which is outside the CpG island and is not normally associated with transcriptional inactivation. However, two samples did have deletions proximal to or within the p16 gene. These results indicate that mutations in p16 may not be a dominant pathway for p16 loss of function or that inactivation of p16 by DNA methylation may not be necessary for the transformation and progression of prostate cancer.