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Roe deer (a seasonal breeder, rut: July to August) is a well characterized model for studying the seasonal regulation of testicular activity. However, not much is known about the impact of estrogens on seasonally determined sperm production. We therefore explored the time and cell type specific expression of estrogen receptors and of enzymes involved in steroid biosynthesis in roe deer testicular parenchyma and in the epididymis. Every second month during the entire seasonal cycle five roe bucks were castrated (n=30). Estrogen receptor (ER) alpha, ERbeta and the enzymes P450Aromatase and P450C17 were localized immunohistochemically. The expression levels of ERalpha, ERbeta and P450Aromatase were evaluated by semi-quantitative Western blot. Contrary to the enzyme required for androgen production (P450C17), which is expectedly located only in the Leydig cells and shows an expression increase towards rutting season, a seasonal expression difference of the enzyme required for the conversion into oestradiol (P450Aromatase) is visible only in the epididymis. In the testis, ERalpha expression shows a striking dependency on tubular cell composition, and the single cell expression activity increases towards rut. This implicates that estrogens are directly involved in the regulation of spermatogenesis in the roe buck. In the epididymis, expression of ERalpha is seasonally determined particularly in the ductuli efferentes. ERbeta was detected throughout the year with no distinct dependency on season or the stages of germinative epithelium cycle. We conclude that estrogens in the roe buck influence the seasonally determined sperm production predominantly by the regulated expression of ERalpha.