A relative low molecular mass bovine kidney acid phosphatase was purified 1,640-fold to homogeneity, with 7% recovery. The purified enzyme (specific activity 100 mumol min-1 mg-1) was electrophoretically homogeneous with a relative molecular mass of 17.8 kDa, as determined by SDS-polyacrylamide gel electrophoresis. A broad pH optimum of 4.0-5.5 and a maximal enzyme activity at 60 degrees C were determined for the p-nitrophenyl phosphate hydrolysis. Apparent Km values of 0.14 mM, 0.4 mM, 0.3 mM and 7.9 mM were obtained, at 37 degrees C and pH 5.0, for the best substrates p-nitrophenyl phosphate, beta-naphtyl-phosphate, flavin mononucleotide and tyrosine-phosphate, respectively. The enzyme activity was enhanced by guanosine but inhibited by ZnCl2 and CuSO4, p-cloromercuribenzoate and ammonium molybdate. Vanadate (Ki 0.47 microM), pyridoxal 5'-phosphate (Ki 2.2 microM), inorganic phosphate (Ki 0.77 microM) are competitive inhibitors. Both glycerol and methanol increased significantly the acid phosphatase activity, acting as good phosphate acceptors in the transphosphorylation reaction.