Short-circuit current (I_sc) technique was used to investigate the role of testosterone in the regulation of chloride secretion in cultured rat efferent duct epithelia. Among the steroids tested, only testosterone, and to a lesser extent, 5-dihydrotestosterone (5-DHT), reduced the basal and forskolin-induced I_sc in cultured rat efferent duct epithelia when added to the apical bathing solution. Indomethacin, a 3-hydroxysteroid dehydrogenase, did not affect the inhibitory effect of 5-DHT. The effect of testosterone occurred within 10-20 s upon application and was dose dependent with apparent IC₅₀ value of 1 µM. The effect was abolished by removal of Cl but not HCO from the normal Krebs-Henseleit solution, suggesting that testosterone mainly inhibited Cl secretion. The efferent duct was found to be most sensitive to testosterone, while the caput and the cauda epididymidis were only mildly sensitive. Cyproterone acetate, a steroidal antiandrogen, or flutamide, a nonsteroidal antiandrogen, did not block the effect of testosterone on the forskolin-induced I_sc, nor did protein synthesis inhibitors, cycloheximide, or actinomycin D. However, pertussis toxin, a G_i protein inhibitor, attenuated the inhibition of forskolin-induced I_sc by testosterone. Testosterone caused a dose-dependent inhibition of forskolin-induced rise in cAMP in efferent duct cells. It is suggested that the rapid effect of testosterone was mediated through a membrane receptor that is negatively coupled to adenylate cyclase via G_i protein. The role of nongenomic action of testosterone in the regulation of electrolyte and fluid transport in the efferent duct is discussed.