Calcium is known to play a crucial role in sperm physiology, including motility, metabolism, acrosome reaction, and fertilization. However, very little is known about the regulation of Ca²⁺ in the epididymis, although it is known that the fluid therein has a lower Ca²⁺ concentration than in the blood plasma. The calcium permeable channel TRPV6, but not TRPV5, is expressed in the reproductive tract of male rats as analyzed by RT-PCR and immunohistochemistry. TRPV6 is predominantly present in the apical membranes of the principal cells of the epididymis and the ciliated cells of the efferent duct. Whole-cell patch-clamp studies of isolated epithelial cells from the rat epididymis revealed a Ca²⁺-selective current with characteristics matching those of the epithelial Ca²⁺ channels, viz constitutive activities, time-dependent inactivation at hyperpolarizing steps of membrane potentials to more negative than –60 mV, inwardly rectifying current-voltage relationship, inhibition by extracellular acidosis but stimulation by alkalosis, and blockade by lanthanum. When the cauda epididymal tubules of anaesthetized rats (pentobarbitone sodium, 60mg kg^-1, i.p. injection) were luminally perfused with HCO₃^–-buffered Krebs solution (pH_o 7.4) in vivo, the perfused segment reabsorbed Ca²⁺ at a rate of 2.6 ± 0.1 nmol cm^-2 min^-1 (n=58 from 16 rats). Reabsorption was dose-dependently suppressed by ruthenium red and lanthanum, putative blockers of epithelial Ca²⁺ channels. Castration markedly reduced the Ca²⁺ reabsorptive capacity of the epididymal tubule (n = 16 rats). This study suggests that TRPV6 provides a Ca²⁺ entry pathway that regulates Ca²⁺ homeostasis in the epididymis. Reabsorption of Ca²⁺ by the epididymal tubule is androgen-dependent.