Until recently, only a single type of estrogen receptor (ER) was thought to exist and mediate the genomic effects of the hormone 17β-estradiol in mammalian tissues. However, the cloning of a gene encoding a second type of ER, termed ERβ, from the mouse, rat, and human has prompted a reevaluation of the estrogen signaling system. Based on in vitro studies, the ERβ protein binds estradiol with an affinity similar to that of the classical ER (now referred to as ERα) and is able to mediate the effects of estradiol in transfected mammalian cell lines. Essential to further investigations of the possible physiological roles of ERβ, and its possible interactions with ERα, are data on the tissue distribution of the two ER types. Herein, we have described the optimization and use of an RNase protection assay able to detect and distinguish messenger RNA (mRNA) transcripts from both the ERα and ERβ genes in the mouse. Because this assay is directly quantitative, a comparison of the levels of expression within various tissues was possible. In addition, the effect of disruption of the ERα gene on the expression of the ERβ gene was also investigated using the ERα-knockout (ERKO) mouse. Transcripts encoding ERα were detected in all the wild-type tissues assayed from both sexes. In the female reproductive tract, the highest expression of ERβ mRNA was observed in the ovary and showed great variation among individual animals; detectable levels were observed in the uterus and oviduct, whereas mammary tissue was negative. In the male reproductive tract, significant expression of ERβ was seen in the prostate and epididymis, whereas the testes were negative. In other tissues of both sexes, the hypothalamus and lung were clearly positive for both ERα and ERβ mRNA. The ERKO mice demonstrated slightly reduced levels of ERβ mRNA in the ovary, prostate, and epididymis. These data, in combination with the several described phenotypes in both sexes of the ERKO mouse, suggest that the biological functions of the ERβ protein may be dependent on the presence of ERα in certain cell types and tissues. Further characterization of the physiological phenotypes in the ERKO mice may elucidate possible ERβ specific actions.