Testosterone (T) synthesised in Leydig cells enters the epididymis and may there be converted into dihydrotestosterone (DHT) by 5α-reductase (5α-red) or into 17β-oestradiol (E2) by P450 aromatase (P450-aro). D-aspartate (D-Asp) is known to induce T synthesis in the testis. In this study, we investigated the effects of in vivo D-Asp administration in two major regions of the rat epididymis (Region I: initial segment, caput, corpus; Region II: cauda). The results suggest that exogenous D-Asp was taken up by both regions of rat epididymis. D-Asp administration induced a rapid increase in T, followed by a more gradual decrease in the T:DHT ratio in Region I. In Region II, T levels rapidly decreased and the T:DHT ratio was consistently lower relative to the control. Expression of 5α-red and androgen receptor genes showed a good correlation with DHT levels in both regions. D-Asp treatment also induced an increase of both E2 levels and oestradiol receptor-α (ERα) expression in Region I, whereas neither E2 levels nor ERα expression were affected in Region II. The early increase of P450-aro expression in Region I and late increase in Region II suggests a direct involvement of D-Asp modulation in P450-aro gene expression. Our results suggest that D-Asp modulates androgen and oestrogen levels and expression of androgen and oestrogen receptors in the rat epididymis by acting on the expression of 5α-red and P450-aro genes.