Prostatic acid phosphatase (EC 18.104.22.168) purified from benign hypertrophic prostate tissue was fractionated by preparative slab isoelectric focusing over a pH gradient of 3.16 to 7.16. Twenty-two of 29 fractions contained enzyme activity. We further examined each active fraction by determining the Michaelis-Menten constant and specific activity. The protein concentration used in the latter determination was estimated either spectrophotometrically or immunochemically by three different radioimmunoassays for the enzyme. Determination of specific activities for each fraction directly correlated enzyme activity with an immunochemical determination, which indicated the immunochemical relationships among different molecular species of the enzyme. We found that the Michaelis-Menten constants for the isolated fractions were similar to the Km value for purified, unfractionated enzyme. Most fractions analyzed by each immunoassay had similar specific activities; the few fractions with discrepant specific activities were found at either end of the pH gradient. The similarity in specific activities among the fractions indicates that RIAs involving polyclonal antisera detect all of the electrophoretic variants of the enzyme.