Urethane‐hydrolyzing activity (urethanase) was found in the homogenates of liver, kidney, and lung of rats. The activity was significantly higher at acidic condition (pH 5.0) than that at neutral condition (pH 7.0) in every case. The highest activity among them was observed in a kidney homogenate preparation. The activities increased slightly with treatment by Triton X‐100. Intracellular distribution of urethanase in rat kidney was examined and found that the enzyme activity located mainly in the lyso‐somal fraction. The optimal pH was found to be 5.0. The enzyme activity was strongly inhibited by EDTA, whose 50% inhibitory concentration was 7.4 × 10−7M. Complete loss of the enzyme activity by the addition of EDTA was fully recovered by the addition of Zn2+, which suggested that urethanase belongs to the category of Zn enzyme.