Aberrant epigenomic alterations include incorrect histone modifications involving altered expression of chromatin-modifying proteins. They contribute to gene silencing and carcinogenesis. The nature of the epigenomic alterations occurring with prostate cancer remains to be fully identified. The acetylation status of histone H3 in human prostate cancer cells was assessed with multiple acetylation sites at N-termini. In contrast to the non-malignant prostatic cell lines RC165N/h and RC170N/h which possess stem cell properties, cancer cell lines LNCaP, DU-145, and PC-3 were either not acetylated or reduced in density (50-70%), at N-termini lysines 9, 14, 18, and 23 of histone H3. Deficient acetylation of histone H3 was similarly detected with clinical prostatic adenocarcinomas as compared to normal tissues. Cancer cell lines and adenocarcinomas exhibited varied acetylation status at particular lysines, indicating the possible presence of deacetylation patterns reflecting individual cancer cell clones. A significantly elevated activity of histone deacetylases (HDACs) was determined in both cancer cell lines and adenocarcinomas. Inhibition of HDACs enhanced histone acetylation and p21 gene expression, indicating that excessive HDAC activity is a requisite for deficient histone acetylation. Deficient histone acetylation involving excessive HDAC activity may represent epigenomic features of prostate cancer cells, and the aberrant enzyme activity is probably an underlying cause of disrupting the epigenomes of normal prostatic cells.