The distribution of acid phosphatases of intermediate molecular weight was determined in various rat tissues. The distribution study of intermediate-molecular-weight acid phosphatase (disignated P-II) in the subcellular fractions of rat liver and kidney indicated that P-II was localized in the mitochondrial fractions. The P-II partially purified from liver showed a pI value of 7.0 on isoelectric focusing, and the apparent molecular weight was estimated to be 40000 by Sephadex G-100 gel filtration or 44000 by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. The enzyme catalyzed the hydrolysis of a wide variety of natural phosphomonoesters, except for phosphoproteins, phosphoserine, o-phosphocholine and thiamine monophosphate. The enzyme showed a high activity on pyridoxal phosphate, β-glycerophosphate, flavin mononucleotide and adenosine 2'-monophosphate. It was markedly inhibited by Hg²⁺ and Ag⁺, but not significantly by sulfhydryl blocking agents or by L-(+)-tartrate.