Lipase [EC 3.1.1.3] was purified from the culture supernatant of Rhizopus japonicus KY 521 by ethanol precipitation, chromatography on Column-lite, affinity chromatography on heparin-Sepharose 4B, and separation into two lipases, I and II, by isoelectric focusing. The purified lipases I and II were found to be homogeneous by disc electrophoresis, and showed isoelectric points at pH 7.4 and pH 7.9, respectively. They both had an apparent molecular weight of about 42,000, hydrolyzed tricaprin very rapidly, and exhibited a pH optimum around pH 7.0-8.5. These lipases were inhibited by the addition of serum to the reaction mixtures. These lipases were enhanced slightly in the absence of serum by high concentrations of NaCl and protamine, but were inhibited strongly by these compounds in the presence of serum.