Estrogens have been implicated in sexual differentiation of both the gonads and the genitalia of birds. In chicken embryos, the gonads are steroidogenically active from an early age, and the aromatase gene, (cAROM), necessary for estrogen synthesis, is expressed only in females at the time of gonadal sex differentiation. However, no studies have directly demonstrated the distribution of estrogen receptor (cER) transcripts or proteins in the embryonic avian reproductive system. Whole-mount in situ hybridization and immunohistochemistry were used here to identify sites of estrogen receptor expression in the embryonic chicken urogenital system. Estrogen receptor mRNA was observed in both male and female gonads prior to morphological differentiation, at Stage 26 (4.5 days of incubation), and continued until after sexual dimorphism at Stage 32 (7.5 days). Transcripts of cER were also detected in the Mullerian ducts and developing external genitalia of both sexes. Estrogen receptor protein was analysed in the embryonic gonads by immunohistochemistry and found to be most abundant in the cortex of the left ovary, although it was also present in the medulla of both female gonads. No significant cER protein expression was detected in the male gonad by immunohistochemistry. In contrast, the aromatase gene was expressed in the gonads of female embryos from the onset of sexual dimorphism but was not detectable in male gonads at any stage examined. These findings suggest that estrogen involvement in both gonadogenesis and genital development in chickens is mediated by the estrogen receptor.