Hyperestrogenemia in humans increases both the concentration of serum T4-binding globulin (TBG) by 2- to 3-fold and the proportion having anodal mobility on isoelectric focusing (IEF). As TBG is synthesized in the liver, we studied the effect of estrogen on TBG synthesis, secretion, and degradation by cultured human hepatocarcinoma cells (Hep G2). beta-Estradiol in concentrations in the range found in pregnancy (10(-7) M) had no effect on the accumulation of immunoreactive TBG in medium over 4 days. The absence of fetal calf serum or phenol red did not alter these findings. The amount of [35S]TBG accumulated 6 h after addition of [35S]methionine was not influenced by exposure to estrogen or to serum obtained from pregnant women. However, 10(-5) M beta-estradiol suppressed TBG more severely than albumin synthesis (34% vs. 9%). The lack of an estrogen effect on TBG synthesis and secretion was supported by experiments showing no effect of estrogen on the disappearance of TBG added to the medium or the accumulation of cytoplasmic TBG mRNA. The same cultures responded to estrogen by a 10-fold increase in nuclear estrogen receptor binding sites and a 2-fold increase in apolipoprotein CII. As TBG in serum, the rate of heat denaturation was not altered in TBG synthesized by Hep G2 cells in the presence of estrogen. In contrast to the effect on TBG in serum, in Hep G2 cells estrogen did not produce an anodal shift on IEF, or increased its proportion not bound to Concanavalin A, nor reduced its clearance rate when injected into rats. However, even untreated Hep G2 cells synthesized TBG with a larger number of anodal IEF bands and proportion of Concanavalin A excluded material than TBG in pregnancy serum. Results support our hypothesis, based on analysis of TBG in pregnancy, that estrogen-induced serum TBG elevation may not be mediated through an increase in synthesis. The failure to observe estrogen induced changes in oligosaccharide structure does not exclude estrogen responsivity of Hep G2 cells. Such effect could be masked by the marked constitutive increase in number of oligosaccharide chain antennae typical in this and other neoplastic tissues.