The effects of estrogen (E2) and progesterone (P) were examined on the expression levels of multiple gap junction (GJ) gene products (alpha 1 = Cx43, beta 1 = Cx32, beta 2 = Cx26) in the uterus and ovaries of immature rats by immunohistochemistry, electron microscopy and northern blot analysis. E2 induced the expression of alpha 1 connexin in the uterus (specifically in the myometrium and in endometrial stroma proximal to luminal epithelium) and ovaries. The E2-induced alpha 1 expression was completely suppressed by P in the uterus, but only partly in ovaries. Steroid hormones also modulated the quantity, size, and distribution of beta 1 and beta 2 containing junctional plaques along lateral cell borders in polarized luminal and glandular uterine epithelia. Small GJs were detected at basolateral regions in proliferative luminal epithelium following administration of E2. In contrast, large GJs were localized at subapical-lateral cell borders of the secretory epithelium following P-treatment. The co-administration of E2 + P had a synergistic effect on beta 1 and beta 2 expression in the luminal epithelium, but an inhibitory effect on beta 2 expression in glandular epithelium. Myometrial GJs were detected in freeze-fracture replicas as aggregates containing regularly arranged particles with particle free zones. In contrast, GJs in secretory epithelium contained particles which were arranged in a non-crystalline fashion. These GJs contained domains of mixed and segregated beta 1 and beta 2 antigens within a single plaque as revealed by laser scanning confocal microscopy analysis of immuno-double-labeled secretory epithelium. The demonstration of segregated antigens within a single GJ plaque indicates the possibility of multiple channel populations formed by homo-oligomeric connexons. These results suggest that different connexins can be differentially regulated by steroid hormones in different cell types, and that the same steroid hormone can have different effects on the same connexin in different cell types.