Advanced hormone-independent prostate cancer is characterized by a significant loss of androgen receptor (AR) expression in 20-30% of the tumors. The transcriptional block underlying this phenomenon is not known, but we have proposed that methylation of CpG sites in the AR promoter may reversibly inactivate transcription of the AR (D. F. Jarrard et al, Cancer Res., 58: 5310-5314, 1998). In this study, detailed methylation analysis using bisulfite sequencing was performed on a series of AR expression-positive and -negative prostate cancer cells. We found that methylation of several consensus sequences in the AR promoter (from -131 to -121 and +44 to +54) are tightly linked to the loss of AR expression in metastatic hormone-independent prostate cancer cell lines. These consensus sites of methylation correlate with the minimal promoter region critical for AR transcription. In human tissues, no methylation was demonstrated in normal or primary prostate cancers that express the AR. Four of 15 tumors obtained from men who had died from hormone-independent prostate cancer demonstrated a significant loss of AR expression immunohistochemically and two (50%) of these AR-negative tumors contained AR methylation. We conclude that the AR promoter contains specific CpG methylation hot spots that are markers for gene silencing. Furthermore, AR methylation may represent a phenotype important in the development of hormone independence in a subset of advanced prostate cancer in which AR expression is lost. The finding of AR methylation also represents the first report of aberrant methylation on an X-linked gene associated with a somatic male cancer.