Epithelial cells were isolated from adult porcine vas deferens and grown in the absence or presence of steroid hormones. Transepithelial resistance (R_te), basal short circuit current (I_sc), and the effects of selected ion transport modulators on these parameters were evaluated in modified Ussing chambers at three time points (5–8, 11–14, and 18–22 days postseeding). At the earliest time point, no significant differences were observed. At the middle time point, when compared with R_te in untreated control monolayers, R_te in monolayers exposed to 17β-estradiol, aldosterone, cortisol, cortisone, prednisolone, prednisone, and dexamethasone was significantly lower; in contrast, R_te in monolayers exposed to testosterone, dihydrotestosterone, or progesterone did not differ from that in control monolayers. Treatments with cortisol, prednisolone, and dexamethasone were associated with an elevated basal I_sc that was amiloride sensitive, indicating ongoing Na absorption by these monolayers. R_te was increased by amiloride treatment in glucocorticoid-treated monolayers but remained significantly less than that of control monolayers. At the third time point, the postamiloride R_te of glucocorticoid-treated monolayers was not different from that of control monolayers. Responses to ATP, forskolin, bumetanide, and DASU-02 were not affected by steroid treatment at any time point. Taken together, these results suggest that estrogens and corticosteroids can modulate epithelial function in the distal excurrent duct of the adult male reproductive system. At physiological or pharmacological concentrations, these hormones would be expected to modify the luminal environment (both the ionic composition and pH) to which sperm are exposed and thus affect male fertility.