The 9 kilodalton vitamin D-dependent calcium-binding protein (CaBP9k), calbindin-D9k, is expressed in the intestine and uterus of mammals. Rat intestinal CaBP9k is a well documented expression of the steroid hormone like action of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). In contrast exogenous 1,25-(OH)2D3 does not affect the concentration of uterine CaBP9k which is dependent on estrogen. We have analyzed the effect of 17 beta-estradiol on the regulation of CaBP9k gene expression in the uterus of mature ovariectomized rats. CaBP9k mRNA is undetectable in the uterus of mature ovariectomized rats. A single dose of 17 beta-estradiol results in a detectable level of CaBP9k mRNA at 1 h and a significant increase 3 h after injection. The maximal CaBP9k mRNA level is reached 6 to 12 h post injection. These results show that 17 beta-estradiol increases CaBP9k production by increasing CaBP9k gene transcription. Chronic 17 beta-estradiol administration results in a plateau of CaBP9k mRNA but in a large increase in CaBP9k concentration. The kinetic response to a single estradiol injection was similar in immature rats. This result shows that no cellular differentiation is required for the control of CaBP9k gene expression by 17 beta-estradiol. The uterine cells of immature rats are already competent to respond optimally to estradiol. There is a single 0.5 kilobase CaBP9k gene transcript in the rat duodenum. In contrast there are two estrogen-inducible CaBP9k mRNA species in the uterus of both the mature ovariectomized and the immature rats. The smallest species corresponds to the duodenal CaBP9k mRNA species, while the larger species is at least 50 nucleotides larger. However, a unique CaBP9k identical to that in the duodenum is expressed in the uterus.