Loss of heterozygosity (LOH) at 6q is a frequent chromosomal aberration in prostate adenocarcinoma; however, a possible target gene remains to be identified. Findings in this study indicate that the FYN tyrosine kinase gene at 6q21 is a new candidate tumor suppressor in prostate cancer. Initially, single nucleotide polymorphism microarray analysis of 40 microdissected prostate adenocarcinoma samples revealed 25% LOH at the FYN locus. Furthermore, Western blot analysis and real-time reverse transcriptase PCR (RT-PCR) showed significantly lower FYN expression in prostate cancer tissue than in benign prostate hyperplasia (BPH), as well as in 6 prostate adenocarcinoma cell lines compared with that in BPH-1 cells. By immunohistochemistry, FYN protein was detected in nonmalignant prostate epithelium, but not in cancerous glands. Moreover, genomic bisulfite sequencing revealed frequent aberrant methylation of a large CpG island in the FYN promoter region in both adenocarcinoma cell lines (3 of 5 cell lines tested) and primary prostate cancer (12 of 18 tumors). Methylation was generally of moderate density, affecting preferentially the 3′ region of the CpG island. Dense hypermethylation of the entire CpG island, consistent with gene silencing, was detected in 2 of 18 tumors (11%). No methylation was found in BPH-1 cells or nonmalignant prostate tissue samples (0 of 7). These results indicate that FYN is downregulated in prostate cancer by both chromosomal deletion and promoter hypermethylation, and therefore is a novel prostate tumor suppressor gene candidate. © 2007 Wiley-Liss, Inc.