In the frog Xenopus laevis, vitellogenin is the major estrogen-induced protein in the liver. We have characterized an additional secreted protein, of 45,000 Da and designated Ep45, which is also responsive to estrogen treatment. Like vitellogenin, Ep45 is not normally found in the plasma nor synthesized by the liver of the male frog. Its synthesis increases 6-fold between days 2 and 8 following a single 2 mg injection of estradiol-17 beta. For comparison, we have also studied a third estrogen-regulated protein, Ep20, with a molecular weight of approximately 20,000. This protein exhibits a different set of characteristics with regard to hormone responsiveness. Ep20 is synthesized in the liver of normal males and therefore is not absolutely hormone-dependent. Its level increases only about 4-fold following estrogen stimulation. The messenger RNAs for both Ep45 and Ep20 have been identified and purified, using a high-resolution RNA fractionation technique. By this procedure, it was possible to demonstrate that following high doses of estrogen the predominant mRNAs in the liver are those coding for vitellogenin, Ep45 and Ep20. Thus estrogen suppression of virtually all other liver proteins appears to act at the messenger RNA level for intracellular as well as secreted proteins.