Arginase (L-arginine amidino hydrolase, EC 3.5.3.1) from cotyledones of Canavalia lineata was purified and characterized. The purification steps involved streptomycin sulfate precipitation, ammonium sulfate fractionation, DEAE-Sephacel ion-exchange chromatography, canavanine-Sepharose 4B affinity chromatography, and Sephadex G200 gel filtration chromatography. Arginase was purified 217-fold with 4% recovery. These column chromatography revealed that ADA (arginine-dependent activity) and CDA (canavanine-dependent activity) eluted the same peak. The pH optimum was 9 for ADA and 8 / or 8.5 for CDA. Kinetic analyses of purified arginase for arginine revealed an apparent K_m of 30 mM at pH 9 and 82 mM at pH 8. Comparable determinations with canavanine revealed an apparent K_m of 62 mM at pH 9 and 43 mM at pH 8. It is suggested that the affinity for canavanine with this enzyme at physiological pH be as high as that for arginine. 1 mM Mn^(2+) was required for the optimal enzyme activity and maximum activity was exerted at 40℃. The molecular weight was estimated as 180,000 by Sephadex G200 gel filtration chromatography and subunit molecular weight was obtained as 44,000 by SDS-PAGE.