Normal prostate, benign prostate hypertrophy (BHP), and prostate adenocarcinoma (ACP) biopsy specimens were analyzed for the presence of human cytomegalovirus (HCMV) and herpes simplex virus type 2 (HSV-2)-specific macromolecules. HCMV DNA homologous sequences were detected in 2 of 13 normal prostate, 2 of 9 BHP, and 3 of 10 ACP tissues, and HSV-2 DNA homologous sequences were found in 1 normal prostate tissue and 2 ACP tissues. In situ DNA-RNA hybridizations indicated HCMV-specific RNA in 3 of 8 BHP and 4 of 9 ACP tissues. No positive in situ hybridization for HCMV RNA was observed in normal prostate tissues. Parallel in situ DNA-RNA hybridization localized HSV-2-specific RNA in only 1 of 8 tumor sections. No HSV-2-specific RNA was observed in sections of normal and BHP tissues. Anticomplement immunofluorescence (ACIF) tests of BHP and ACP specimens showed specific HCMV immunofluorescence in 3 of 8 BHP and 4 of 9 ACP tissues. ACIF results correlate closely with in situ hybridization results and imply some degree of HCMV association with prostatic abnormality. The results also suggest that latent HCMV may be harbored by the human prostate gland.