Differences in the responses to glucocorticoids and pregnenolone 16 alpha-carbonitrile (PCN) of three isozymes of hepatic microsomal carboxylesterase, namely RL1, RL2 and RH1, in male rats were studied. The administration of dexamethasone dose-dependently increased isocarboxazid hydrolase activity, whereas p-nitrophenyl acetate-hydrolyzing activity was decreased dose-dependently. Betamethasone, methylprednisolone and PCN also markedly increased isocarboxazid hydrolase activity. A radial immunodiffusion assay indicated that carboxylesterase reactive with antibodies was induced by these steroids. Carboxylesterase isozyme RL2 was strongly induced by dexamethasone, methylprednisolone and PCN. In contrast, RL1 and RH1 were decreased by dexamethasone, but not by the other steroids. Estradiol benzoate had a synergic effect on the PCN-induced increase of isocarboxazid hydrolase, but the actions of the glucocorticoids were not affected. It is concluded that hepatic microsomal carboxylesterase isozymes in rats differ considerably from each other in their response to various steroids. These data are also indicative of the importance of glucocorticoids in hepatic xenobiotic metabolism.