The induction of long-term responses in the uterus following estrogen treatment is discussed, with special reference to DNA synthesis. Immature female rats injected daily with estradiol-17beta or estriol (0.01 to 1 mug) or a combination of the two steroids for one, two or three consecutive days were sacrificed at intervals from 12 to 24 h after the last injection of vehicle or steroid. In vitro incorporation of [3H]thymidine into DNA [14C]leucine into protein, and oxidation of [14C)glucose to 14CO2 were determined. Nuclear-bound estradiol was determined by use of exchange assay or following incubation of intact uteri with 1 X 10(-8)M ([3H]estradiol for 1 h at 37 C. Injection of estriol only partially stimulated DNA synthesis by 18 to 24 h post-treatment. However, injection of estriol followed by injection of estradiol 6 h later resulted in increased DNA synthesis, suggesting that estrogen must be present for up to 6 h to induce subsequent DNA synthesis. Maximal DNA and protein synthesis and oxidation of glucose occurred at 24 h after injection of estradiol (0.1 or 1 mug) but was depressed to control levels by 24 h after the last of three daily injections. Daily injections of 0.01 mug of estradiol resulted in a similar pattern of DNA synthesis, although of lesser magnitude than that observed after injection of 0.1 or 1 mug of estradiol. However, if rats receiving daily injections of 0.01 mug estradiol were challenged with a higher dose of estradiol (1 mug), uterine DNA synthesis was markedly increased. The data suggest that prolonged exposure to estrogen causes uterine cells to become metabolically "refractory" to further estrogen stimulation. Sequential injections of estriol (1 mug) or intermittent injections of estradiol (1 mug) were ineffective in causing this uterine "refractoriness". Receptor binding of estrogen, translocation to the nucleus, and retention of receptor in the nucleus were not affected by sequential estrogen treatment. The accumulation of an inhibitory product is suggested as a possible explanation for this phenomenon.