Prostate specific antigen, the clinical marker for prostate cancer, is a neutral serine protease whose function is to lyse seminal proteins. Recent work by our laboratory has suggested that prostate specific antigen stimulates the generation of reactive oxygen species in prostate cancer cells. Using 2',7'-dichlorofluorescin diacetate, a dye that fluoresces in the presence of hydrogen peroxide or hydroxyl radicals, we found that prostate specific antigen markedly stimulated reactive oxygen species generation in LNCaP cells. The effect was concentration dependent and its specificity was supported by the fact that anti-prostate specific antigen antibodies abolished the response. Since testosterone stimulates the production of prostate specific antigen, we considered that the reactive oxygen species response to testosterone may be linked to prostate specific antigen. We found that the testosterone effect on reactive oxygen species was blocked by flutamide and by anti-prostate specific antigen antibody. Additionally, though PC3 and DU145 could not respond to testosterone, they readily increased reactive oxygen species in response to prostate specific antigen. Focusing on the mechanism of the prostate specific antigen effect, we tested two other serine proteases, trypsin and chymotrypsin, but found no effect on reactive oxygen species in LNCaP cells. Nevertheless, serine protease inhibitors, alpha(1)-antichymotrypsin, alpha(2)-macroglobulin and Bowman-Birk inhibitor, blocked reactive oxygen species generation stimulated by prostate specific antigen. This apparent paradox was investigated with the use of a specific anti-'prostate specific antigen' antibody which recognizes an epitope away from the catalytic site and which does not inhibit protease activity. Despite the lack of inhibition of proteolytic activity, this antibody blocked the effect of prostate specific antigen on reactive oxygen species generation. These findings suggest that although the integrity of the prostate specific antigen molecule is necessary for stimulating reactive oxygen species generation, its proteolytic activity is not. The underlying mechanism is currently under investigation.