The tissue distribution of cocaine methyl esterase and ethanol-dependent ethyl transferase activities was determined in the rat and compared to the tissue distribution of three distinct non-specific hydrolases. Rates of formation of benzoylecgonine from cocaine and cocaethylene from ethanol and cocaine were measured in serum and tissue homogenate-supernatants of the brain, heart, kidney, liver, lung and spleen. The tissue distribution of three nonspecific esterases, A, B and C, was defined by nondenaturing gel electrophoresis and measuring the hydrolysis of 4-methylumbelliferyl acetate in the gels. Immunoreactive protein was localized by using Western blot analysis with polyclonal rabbit antihuman liver cocaine methyl esterase antibody after denaturing and nondenaturing gel electrophoresis. The rat liver, lung, kidney and heart exhibited cocaine methyl esterase and ethyl transferase activities and immunoreactive protein. The brain had cocaine methyl esterase activity but no ethyl transferase activity; neither activity was found in serum or spleen. The dominant immunoreactive bands in the liver, lung, kidney and heart comigrated with the 59 kD band of purified human liver cocaine methyl esterase. The rat liver, lung and kidney exhibited a band of nonspecific esterase activity that migrated with purified human liver cocaine methyl esterase and rat hydrolase A. These observations suggest that rat hydrolase A is similar to human cocaine methyl esterase. The lack of straight forward correlation between cocaine methyl esterase activity and immunoreactive protein and nonspecific esterase activity suggests that more than one enzyme catalyzes the hydrolysis of cocaine to benzoylecgonine in the rat.