Bioelectrical properties and anion secretion in cultured epithelia from different regions of rat and human male excurrent ducts were studied by measuring the short-circuit currents (ISC). In all regions of the rat excurrent duct, Cl- secretion accounts for over 90% of the basal ISC, although the magnitude varied in different regions. Cl- secretion was found to be mediated by a Cl-/HCO3- exchanger, an Na+/H+ exchanger, and an Na+/K+/2Cl- symport located on the basolateral side of the epithelial cells. Forskolin, an activator of adenylate cyclase, and ionomycin, a Ca2+ ionophore, were used to investigate the relative importance of cAMP and Ca2+ as intracellular messengers regulating Cl- secretion in different regions. It was found that in both species, the forskolin-evoked ISC response was larger in the proximal end (efferent duct/caput epididymidis [rat/human, respectively]) than in the distal end (cauda/corpus epididymidis). The response to ionomycin in the rat cauda epididymidis (distal end) was larger than that in the efferent duct (proximal end); on the other hand, no significant difference in the ionomycin-induced ISC was observed in the caput and the corpus regions from the human epididymis. Our results indicate that while the cAMP- and Ca(2+)-dependent pathways are both involved in regulating Cl- secretion in all regions along the male excurrent ducts in both species, a regional difference exists with respect to the relative importance of the two regulatory pathways involved in Cl- secretion along the male reproductive tract.