NKX3.l is a homeoprotein with prostate-specific expression in adults. Loss of NKX3.l correlates with prostate cancer progression. Loss of heterozygosity affects NKX3.l in about 80% of prostate cancers. This project focuses on DNA methylation of the NKX3.l gene promoter in prostate cancer cell lines and tissues. DNA was analyzed by methylation- specific PCR and sequencing of bisulfite-treated DNA. We also treated cultured cells with the methylation inhibitor 5-azacytidine and the histone deacetylase inhibitor, trichostatin A. There was no effect of 5-azacytidine or trichostatin A on NKX3.l expression in cultured cell. By bisulfite modified DNA sequencing, we identified some methylated or partial,methylated CpG islands in -1056 to 1172 of NKX3.l gene. Three CpG sites at -921, -903 and -47 were selectively methylated in different prostate cancer cell lines and were also methylated in some prostate cancer tissue. Methylation in tissue correlated inversely with NKX3.l protein expression. We also study the effect of Spl and Sp3 transcription factors on the expression of NKX3.l, our results showed Spl and Sp3 did not influence on the expression of NKX3.l.