Two L-asparaginase fractions have been isolated from the cytosol (105,000 g supernatant)of guinea pig liver, L-Asparaginase I has been purified 53-fold by sodium sulfate precipitation,gel permeation chromatography, ion exchange chromatography and negative adsorption from calcium hydroxylapatite. As judged by polyacrylamide gel disc electrophoresis and sedimentation velocity ultracentrifugation,this enzyme is homogeneous. The Svedberg constant for L-asparaginase I is 13.10 X 10^-13 s and for Sephadex purified L-asparaginase II, 9.24 x 10^-13 s. The pH optimum of L-asparaginase I is 8.6-9.0 and of L-asparaginase II, 7.2.-7.6. The K(m) and V(max) for L-asparaginase I were different when compared to the same values for L-asparaginase II. Inhibition of L-asparaginase I by glycine has been noted.