Comparison of zymograms of acid phosphatases (orthophosphoric monoester phosphohydrolase, acid optimum, E.C.3.1.3.2) from human prostate, leukocytes, seminal vesicles, and seminal fluid, separated by analytical isoelectric focusing (IEF), resulted in the identification of three individual groups, particularly of the prostate. These groups contain three molecular forms at different molecular weights and activities in varying quantities and combinations. The molecular weights, estimated by SDS gel electrophoresis, were 1) 86,000, 2) 76,000, and 3) 46,000/50,000 (in a doublet) daltons. None of these isoenzymes were restricted to the prostate, but they were present in very high concentrations in the prostate. Compared to the prostate, the seminal vesicles and isolated leukocytes had very closely related zymograms of acid phosphatases. No specific inhibitor has been found that would selectively inhibit one particular isoenzyme without affecting the others. Protein titration and staining activity of IEF gels at different pH values showed that the isoenzymes from all three groups have high hydrolytic activity of orthophosphoric monoesters beyond the acidic pH range of pH 4-5. Incubation of acidic isoforms of acid phosphatases with neuraminidase did not result in the formation of a homogeneous stem molecule. Further analysis of the secretory moiety using the western blotting method showed a binding of peroxidase-conjugated concanavalin A (Con A), indicating that these isoenzymes are glycoproteins. Moreover, isoenzymes extracted from prostate, seminal fluid, and human leukocytes are immunologically identical.