The gender-specific differences in the content of cocaine methyl esterase and ethyl transferase activities are examined in rat tissues and related to differences in hydrolase A protein in rat liver, lung, and kidney reported previously. The rat hydrolase A catalyzes the conversion of cocaine to benzoylecgonine and the ethyl transesterification of cocaine to form cocaethylene. An HPLC assay was used to quantitate and compare cocaine esterase activities in male and female rat tissues. The cocaine methyl esterase and ethyl transferase activities are 1.4 to 2.5 fold greater in male than in female liver and slightly greater in female than in male lung. No gender-specific differences were detected in the kidney. Gel electrophoresis was used to separate three non-specific carboxylesterases (hydrolases A, B, and C) in rat tissues and the isoenzymes were visualized with a hydrolase activity stain using 4-methylumbelliferyl acetate as substrate. The activity of cocaine methyl esterase and content of hydrolase A protein are not consistently different in the lung or the kidney of male versus female rats. Activity of hydrolase A in gels of male liver is greater than in female liver. Similarly, the content of the corresponding hydrolase A immunoreactive protein in male liver is 1.6 fold greater than in female liver. In contrast to hydrolase A, hydrolase C activity is greater in gels of female than male liver extracts. The greater content of cocaine methyl esterase and ethyl transferase activity in male versus female rat livers suggests that there may be gender-specific differences in pharmacokinetics of cocaine metabolism and extent of cocaine-induced hepatotoxicity in rats.