By the use of ribonuclease protection assay (RPA) combined with immunohistochemical techniques, the expression of estrogen receptor (ER) α and ERβ was mapped in the developing gonads and reproductive tracts of male and female mice from fetal day 14 to postnatal day 26 (PND 26). This study was designed to determine the pattern of expression of both ER subtypes in specific tissue compartments during development. In ovaries, ERα mRNA was detected at all ages examined; ERβ mRNA was seen as early as PND 1, and its expression increased with age. Immunolocalization showed ERβ in differentiating granulosa cells of the ovary, whereas ERα was predominantly seen in interstitial cells. The remainder of the female reproductive tract showed ERα mRNA at all ages examined with little or no significant levels of ERβ, except on PND 1 when a low level of message appeared. In males, ERα and ERβ mRNA were detected in the fetal testis; however, ERβ gradually increased until PND 5 and subsequently diminished to undetectable levels by PND 26. Immunolocalization showed ERα in the interstitial compartment of the testis, whereas ERβ was seen predominantly in developing spermatogonia. The remainder of the male reproductive tract showed varying amounts of both receptors by RPA and immunostaining throughout development. These studies provide information useful in studying the role of both ER subtypes in normal differentiation, and they provide indications of differential tissue expression during development.