Transcriptional silencing of antitumor genes via CpG island methylation could be a mechanism mediating prostate cancer (PCa) progression from an androgen-sensitive (AS) to an androgen-insensitive (AI) state. We have used the methylation-sensitive restriction fingerprinting (MSRF) technique to identify novel CpG-rich sequences that are differentially methylated between the genome of the AS PCa cell line LNCaP and that of an AI subline LNCaP(CS) generated by maintaining LNCaP in medium with charcoal-stripped (CS) serum for over 30 passages. One such sequence identified was located on a 5' CpG island that was found to span part of the promoter, exon 1, and part of intron 1 of the peroxisomal membrane protein 24 kDa (PMP24) gene. Using semiquantitative RT-PCR and bisulfite genomic sequencing, we established an inverse relationship between mRNA expression and methylation of the 5' CpG island of PMP24. PMP24 mRNA was absent in LNCaP(CS) and the androgen receptor-negative PC-3 cell line; both exhibited dense methylation in the said CpG island. In contrast, PMP24 mRNA was expressed in LNCaP and normal prostatic epithelial cells (NPrECs) whose PMP24 5' CpG island remained unmethylated. Treatment of LNCaP(CS) and PC-3 with the demethylating agent 5-aza-2'-deoxycytidine (5-AZAdC) reactivated PMP24 mRNA expression. Transient transfection of PMP24 into LNCaP(CS) and PC-3 cells induced a significant reduction in cell growth and soft-agar colony formation potential, suggesting that PMP24 gene product has antitumor properties. These results demonstrate the utility of MSRF in the identification of novel, differentially methylated DNA sequences in the genome and suggest that hypermethylation-mediated silencing of PMP24 is an epigenetic event involved in PCa progression to androgen independence.