The rate of in vitro production of thyroxine-binding globulin (TBG) was studied in hepatocytes isolated from 6 control rhesus monkeys (serum TBG: 19.6 +/- 0.5 micrograms/ml; mean +/- SE) and 6 monkeys treated for 4-5 weeks with beta-estradiol (E2) (serum TBG: 45.1 +/- 1.8 micrograms/ml). Incorporation of [3H]leucine into intracellular soluble and particle-bound TBG, and into secreted TBG was determined for incubation periods up to 9 h. TBG was purified by affinity chromatography and measured by specific immunoprecipitation. The absolute amount of [3H]TBG and the ratio of [3H]TBG to total labeled protein in the same fraction were 3-fold higher in the particulate fraction and in the incubation medium of hepatocytes isolated from E2-treated monkeys. In separate experiments, TBG accumulation in the medium was measured for periods up to 19 h by radioimmunoassay. A 2.4-fold increase was observed with hepatocytes from E2-treated monkeys (3.48 ng TBG/h/10(7) cells, compared to 1.46 in controls). Correction of the production rates for the number of cells surviving during the incubation, and assuming 10.2 x 10(9) cells per liver, gave TBG production rates of 250 micrograms/liver/day in hepatocytes from E2-treated monkeys and 104 micrograms/day in hepatocytes from control monkeys. These experiments demonstrate that estrogen increases in vitro synthesis and secretion of TBG by isolated hepatocytes. The observed 2.4 to 3-fold increase was similar to the 2.9-fold increase in TBG production measured in vivo by kinetic analysis of TBG metabolism.