Plasma pseudocholinesterase (PsChe) activity was examined in adult female rat hepatocytes isolated by collagenase perfusion and maintained in a chemically defined medium supplemented with dimethyl sulfoxide. Time course studies on PsChe activity in cultured hepatocytes indicate that cells maintained in a chemically defined medium lacking human GH and 17 beta-estradiol (E2) exhibit a decrease in activity after the first 3 days in culture followed by a stabilization of PsChe activity for up to 15 days. GH (0.02, 0.2, and 2 micrograms/ml) increased PsChe activity in a dose-dependent manner. Addition of E2 (10(-5)-10(-7) M) alone to hepatocyte cultures did not cause an increase in PsChe activity. The increases produced by both the 2 micrograms/ml and 0.2 micrograms/ml GH doses plus E2 (10(-7) M) were significantly greater than controls and similar to the increase produced by GH alone. The ability of the hepatocytes to express PsChe activity was not dependent upon the continuous exposure of the cells to GH, since control cultures, maintained for 12 days in medium lacking GH, were able to express a high level of PsChe activity after the addition of GH (2 micrograms/ml) on day 12. This increase was observed in hepatocytes in culture for 30 days. These results indicate that GH plays a pivotal role in the regulation of PsChe activity in vitro, and that under the conditions used in this study, E2 does not influence the ability of hepatocytes in culture to express this enzyme.