DEAE-Sephadex chromatography of male rat urine resolved three peaks of arginine esterase activity using the synthetic substrate alpha-N-p-tosyl-L-arginine methyl ester . HCl (Tos-Arg-O-Me). Esterase activity in peak 1 (esterase A1 fraction) was present in sexually mature males, but it was not found in mature females, castrated mature males, or sexually immature males. Urinary esterase A1 activity could be restored in castrated mature males by the administration of testosterone, and it is, therefore, androgen dependent. Esterase A1 activity was undetectable and could not be induced in females by daily doses of testosterone (up to 20 mg/day). The second urinary esterase peak (esterase A2 fraction) and the third peak (urinary kallikrein) were found in both male and female rats. Molecular sieve chromatography of esterase fractions A1, A2, and kallikrein, as determined by Sephacryl S-200 chromatography, gave single peaks of activity. Molecular weights were estimated to be 28,500, 42,000, and 41,500, respectively. Kinin-generating activities of esterases A1, A2, and kallikrein were also determined using dog plasma substrate. Esterase A1 fraction had no kinin-generating activity, while esterase A2 fraction demonstrated significant activity but was 12 times less active than kallikrein per Tos-Arg-O-Me esterase unit. Esterase A2 fraction could not induce direct contraction of the rat uterus, and A2 did not cause a transient decrease in rat blood pressure when injected iv, both of which are criteria for kallikrein activity. It is concluded that the androgen-dependent Tos-Arg-O-Me esterase has properties quite different from both esterase A2 and kallikrein. Esterase A2 appears to be similar to the esterase A described in female rat urine by Nustad and Pierce.