Estrogens stimulate DNA synthesis and cell proliferation in the uterus. All major uterine cell types (luminal and glandular epithelium, stroma, and myometrium) respond to 17 beta-estradiol in the immature animal, whereas primarily epithelial cells of the uterine endometrium respond in the mature animal. Rapid activation of the c-fos protooncogene by estrogen precedes the uterine growth, suggesting that c-fos plays a role in amplifying the hormonal signal. The specific uterine cell types in which estrogen induces c-fos messenger RNA (mRNA) expression, however, have not been identified in either mature or immature animals. In this study, in situ hybridization was used to determine the cell type-specific location of mRNA encoding c-fos in the uterus. In both immature and mature castrated rats at 3 h after 17 beta-estradiol administration, c-fos expression was detected primarily in uterine luminal and glandular epithelia. Expression of c-fos returned to baseline levels by 24 h post 17 beta-estradiol treatment. There was no apparent difference in the uterine cell type-specific pattern of c-fos expression stimulated by estradiol in mature vs. immature animals. Nuclear run-on transcription assay in isolated luminal epithelial cell nuclei showed that c-fos gene transcription increased rapidly in the uterus after estradiol stimulation. Treatment of adult rats with a single injection of 16 alpha-estradiol, a short-acting, nonmitogenic estrogen, induced c-fos primarily in the uterine glandular epithelia. Progesterone is known to modify the action of estrogen on the uterus by redirecting the proliferative response from epithelia to stroma. To determine if progesterone modulation of estrogen action involves shifting of c-fos expression to stromal cells, rats were treated with progesterone for 48 h and then killed 0, 3, 6, or 12 h after an estradiol injection. In situ hybridization analysis revealed that c-fos mRNA remained localized in the uterine luminal and glandular epithelia, and expression was not shifted to the stroma. Although these results support the idea that c-fos plays a role in proliferation of uterine epithelial cells, they also invite reassessment of the role played by c-fos in both epithelial and nonepithelial uterine cell types.