The binding of estrogen to alpha-fetoprotein (AFP) in the plasma cannot account for the impaired estrogen response seen in immature rodents because estradiol (E2) doses that far exceed the total body burden of AFP will stimulate only modest uterine growth. We investigated this phenomenon in immature female mice by determining their uterine weights 23 hr after intraperitoneal injection of estrogens or AFP or both. Administration of either 0.5 micrograms of E2 or 10 ng of moxestrol (MOX) approximately doubled the uterine weight. Giving 1 microgram of AFP 1 hr before injection of either estrogen did not alter that response. Combining the E2 and AFP just prior to injection resulted in decreased uterine growth (34% inhibition). Preincubating the estrogens with purified AFP (0.1-50 micrograms) did not affect the growth response to moxestrol but markedly decreased the response to E2. This was not due to sequestering of hormone because maximal reduction of the E2 response (ca. 65% inhibition) required only 1.0 microgram of AFP (AFP/E2 molar ratio, 1:130), and higher AFP doses inhibited less. About 40% of the growth elicited by injection of either 0.5 micrograms of E2 or 10 ng of MOX was inhibited when these doses were preceded by injection of the preincubated AFP/E2 mixture but not when preceded by either of the components. In each experiment, the mitotic index of luminal epithelium was affected to the same degree as uterine weight. AFP and E2 incubated for 1 hr thus produce a potent inhibitor of estrogen-stimulated mitotic activity and growth. This inhibitor might act upon estrogen-responsive cells at specific sites at which competition by an inactive component of AFP can block the process.