A phosphatase hydrolyzing 3-phosphohistidine and 6-phospholysine was purified from rat brain cytosol to 90% homogeneity on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. One milligram of protein of the purified phosphatase released inorganic phosphate from 3-phosphohistidine, 6-phospholysine, AMP, GMP, and p-nitrophenyl phosphate at velocities of 6.5, 15.6, 15.0, 6.9, and 8.3 mumol/min, respectively. However, the purified enzyme could not hydrolyze N omega-phosphoarginine and phosphocreatine, which are substrates for phosphoamidase [EC 3.9.1.1]. The molecular masses of the holoenzyme and the subunit were 94 and 50 kDa, respectively, and the sedimentation coefficient of the native enzyme was 6.3 s, indicating that it was a dimeric enzyme of identical subunits. The enzyme functioned well under acidic conditions, and 50% of the activity was inhibited by 30 microM tartrate, 4 microM vanadate, 20 microM molybdate, 4 microM VCl3, or 13 microM MoCl5. These results indicate that the present hydrolase belongs to the acid phosphatase group [EC 3.1.3.2].