The minor enzyme of human prostatic acid phosphatases (pI 5.5) with high specific activity (orthophosphoric monoester phosphohydrolase, acid optimum, EC 3.1.3.2) has been purified for the first time as a pure enzyme protein. The enzyme was a single protein when examined by polyacrylamide gel electrophoresis and isotachophoresis. The specific activity was 1080 micromole per (min X mg) for hydrolysis of 5.5 mmole per liter of p-nitrophenylphosphate at pH 4.8 and 37 C. The purification coefficient was 540 and the recovery of enzyme activity was 2 per cent. The molecular weight of the enzyme subunit when measured by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate was 54,000. The Km of the purified enzyme was 3 X 10(-4) mole per liter for p-nitrophenylphosphate. An antiserum to this enzyme was prepared. The enzyme was cross-reactive with the main enzyme (pI 4.9) of human prostatic acid phosphatases in immunoelectrophoresis. No precipitin arc with the acid phosphatase in the serum of a prostatic carcinoma patient could be shown. Antiserum to the main enzyme caused a precipitin line with the same serum sample.