Nitric oxide (NO) is an important signaling molecule for ischemia, inflammation, angiogenesis, immune response, and cell growth and differentiation. It has recently been shown that increased production of NO within various human cancers may contribute to tumor angiogenesis, tumor growth and metastasis, and tumor-related immune suppression. NO can be produced by several NO synthases (NOS), including inducible synthase (iNOS), which is expressed during cell activation and produces NO in larger quantity and for a longer period of time than non-inducible NOSs. In this study, we examined the expression levels of iNOS mRNA and protein in prostate adenocarcinoma using a paired nonneoplastic and neoplastic primary prostate cell culture system and related prostatectomy specimens. Six pairs of neoplastic and nonneoplastic primary prostate cell cultures were established from radical prostatectomy specimens based on homogeneity of the originating tumor and the nonneoplastic tissue. Radioactive reverse transcriptase polymerase chain reaction and subsequent quantitative analysis of iNOS mRNA were performed on the cultures using beta-actin as an internal control. Immunohistochemical studies with an anti-iNOS monoclonal antibody were performed on the corresponding formalin-fixed paraffin-embedded prostatectomy tissue sections. We observed marked patient-to-patient variation in "normal" levels of iNOS mRNA. However, all six neoplastic cultures showed moderately to markedly higher mRNA levels than did their paired nonneoplastic cultures. In addition, iNOS protein levels were significantly higher in paraffin-embedded prostate cancer tissue sections than in adjacent nonneoplastic tissue. Overexpression of iNOS mRNA and protein levels is present in moderately differentiated prostate adenocarcinoma and may contribute to prostate cancer angiogenesis, tumor growth, and tumor-related immunosuppression.