Background: Prostate cancer development is initially steroid hormone dependent. Estrogen receptors (ERs), androgen receptors (ARs), and progesterone receptors (PRs) have been identified in normal and cancerous prostate tissues. We investigated whether the promoter regions of these steroid receptor genes are methylated and inactivated in prostate cancer cells and tissues. Methods: The expression and promoter methylation status of three ERα isoforms (ERα-A, ERα-B, and ERα-C), ERβ, two PR isoforms (PR-A and PR-B), and AR were investigated in five prostate cancer cell lines (ND1, DU145, PC3, LNCaP, and DUPro) and in pairs of normal and cancerous prostate tissues from 38 patients with prostate cancer. Methylation-specific polymerase chain reaction, reverse transcription–polymerase chain reaction, and 5` rapid amplification of complementary DNA ends were used. All statistical tests were two-sided. Results: ERα-C was expressed in all cell lines, but ERα-A and ERα-B were not expressed in any cell line. ERα-A and ERα-B promoters were methylated, but ERα-C was unmethylated. Promoters for ERβ, AR, PR-A, and PR-B were methylated and thus inactivated in some cell lines but not in others. Treating cells with the demethylating reagent 5-aza-2`-deoxycytidine restored expression of all steroid receptor genes with previously methylated promoters. All 38 pairs of cancer and normal tissues had unmethylated ERα-C promoters. Thirty-six (95%) of 38 cancers had methylated ERα-A, 35 (92%) of 38 cancers had methylated ERα-B, but all normal tissues had unmethylated ERα-A and ERα-B (both P<.001). ERβ was methylated in 30 (79%) of 38 cancers but unmethylated in all normal tissues. AR was methylated in three (8%) of 38 cancers but unmethylated in all normal tissues. PR-A and PR-B were unmethylated in all tissues. Conclusion: Certain steroid receptor genes appear to be inactivated by CpG methylation in prostate cancer tissue and cell lines.