Androgens and estrogens regulate epididymal function but the mechanisms by which these hormones act is not fully understood. Epididymal culture systems have been described but none of these identify if AR, ERα and ERβ are expressed concurrently under identical culture conditions. Presumably, the actions of androgens and estrogens require their receptors and our results demonstrate for the first time that rat epididymal cell cultures express AR, ERα and ERβ protein under identical culture conditions. Furthermore, we demonstrate that the expression of these receptors in vitro mirrors normal in vivo expression patterns, a key finding for past and future studies. An epididymal culture system that maintains expression of androgen and estrogen receptors will allow for future investigations into the regulation and function of the epididymis. Previous studies showing prepubertal expression of ERα, did not find ERα expression in adult animals, making our study the first to demonstrate both prepubertal and adult expression of ERα. Additionally, species differences have been suggested to exist with regards to epididymal expression of ERα. Our results are the first to experimentally compare ERα expression in two different rat species and show that expression is similar between the two species. The expression of ERα and ERβ protein prior to puberty and into adulthood provides further supports for the hypothesis that the epididymis may be influenced by estrogens, in addition to androgens, during development and mature function.