Previous reports have shown elevated arginase activity in prostate cancer patients. This study was designed to compare expression levels of arginase II (AII) in various human prostate cancer cell lines and tissues. Expression levels of AII and other enzymes involved in arginine metabolism were examined in androgen-dependent (LNCaP, LAPC-4) and androgen-independent (PC3, DU145, CL-1, CL-2) prostate cancer cell lines by real-time RT-PCR and Western blot analysis. Further expression analysis of AII was accomplished by immunohistochemical staining of a tissue microarray comprised of 246 primary prostatectomy specimens. In addition, polyamine levels were measured within the prostate cancer cell lines by HPLC. Total polyamines were significantly lower in the androgen-dependent cell lines compared to the androgen-independent cell lines. AII expression was found to be most prominent in the androgen-dependent cell lines and least prominent in the androgen-independent cell lines. Additionally, we found expression of ornithine aminotransferase (OAT), an enzyme also responsible for ornithine production, to be inversely correlated with AII expression. The tissue microarray data revealed that the highest AII expression was seen in BPH, followed by PIN and normal samples, with the lowest expression levels observed in prostate cancer tissues. Moreover, we observed an expression gradient across Gleason grades revealing stronger AII expression in low-grade tumors. The polyamine data, combined with the expression analysis studies, support a possible correlation between AII, OAT, and polyamine synthesis. Based on these results, arginase II expression may play a role in prostate cancer progression. More specifically, the elevated AII expression seen in androgen-dependent and in more differentiated prostate cancers suggests that AII could be a potentially useful marker of early stage prostate adenocarcinoma.