The present study used in situ hybridization histochemistry to compare the distribution of estrogen receptor (ER)-alpha and ER-beta mRNA-containing cells in rat pituitary, gonads, uterus, and prostate of intact animals or after hormonal manipulations. Cryostat tissue sections were hybridized with 35S-labeled antisense riboprobes complimentary to ER-alpha or ER-beta mRNA, stringently washed and apposed to emulsion. The results of these studies indicate that the expression of the two receptors is tissue and region specific, with estrogen target tissues specifically expressing ER-alpha, ER-beta, or both forms of ER. In the intact rat, ER-alpha and ER-beta mRNA were both seen in the pituitary, although more cells expressed ER-alpha than ER-beta mRNA. The distribution of the two transcripts in the ovary was qualitatively different, with ER-alpha being primarily localized in the stromal cells, while ER-beta mRNA was concentrated in the granulosa cells of developing follicles. In the uterus, ER-alpha mRNA was abundant in the stromal and epithelial cells of the endometrium, while only very weak ER-beta hybridization signal was detected in these cells. ER-beta mRNA-expressing cells, but not ER-alpha, were also detected in the prostate and in the Sertoli cells, and the large, round spermatocytes of the testis. Gonadectomy markedly attenuated the expression of ER-beta mRNA in the peripheral tissues, with the level of ER-beta mRNA in the uterus and prostate reduced to non-detectable levels. The results of these in situ hybridization studies demonstrate that the distribution and regulation of ER-beta mRNA expression is tissue specific and different from ER-alpha mRNA. The differential expression of ERs in these tissues may explain in part the tissue selective activity of estrogenic compounds.