Synthesis of uteroglobin in rabbit uterine epithelial cells during early pregnancy is induced by progesterone and apparently suppressed by estrogen. To assess whether the steroid hormones exert regulatory effects on the expression of the uteroglobin gene, we performed transcriptional rate assays in endometrial nuclei prepared from ovariectomized rabbits subjected to different hormonal treatments. The isolated nuclei were incubated with [3H]UTP to assess the synthesis of newly elongated RNA transcripts by the incorporation of [3H]UMP. The percentage of labeled RNA represented by uteroglobin sequences was determined by hybridization with cloned uteroglobin DNA immobilized on nitrocellulose filters. Progesterone alone (3 mg/kg X day) for 5 days increased endometrial DNA content 16-fold, total cellular transcriptional activity 4-fold, and relative uteroglobin gene transcription about 6-fold. 17 beta-Estradiol alone (50 micrograms/kg X day) for 5 days had no significant effect on any of these three variables. Not until after 2 days of treatment did progesterone increase the relative rate of uteroglobin gene transcription, whereas total transcriptional activity per mg DNA increased rapidly on the first day of progesterone administration. Treatment of progesterone-stimulated rabbits with estradiol for 5 days reduced tissue DNA content by 80%, and a single injection of estradiol caused a small decrease in uteroglobin gene transcription. Chronic treatment with progesterone alone was sufficient to reduce uteroglobin gene transcription to control levels, without a decrease in tissue DNA content. Progesterone thus regulates uteroglobin synthesis in the uterus at least at three levels: 1) by exerting a mitogenic effect on endometrial cells, 2) by enhancing total cellular transcriptional activity, and 3) by preferential stimulation of uteroglobin gene transcription. Physiologically, the decline in uteroglobin synthesis after 5 days of pregnancy appears to be the result of a decrease in uteroglobin gene transcription in the face of the continued rise in blood progesterone, and this effect may be aided by antiprogestational actions of estradiol.