Neurotransmitter-controlled Cl⁻ secretions play an important role in maintenance of the epididymal microenvironment for sperm maturation. This study was carried out to investigate the effect of carbachol (CCH) on the cultured rat epididymal epithelium and the signal transduction mechanisms of this response. In normal K-H solution, CCH added basolaterally elicited a biphasic Isc response consisting of a transient spike followed by a second sustained response. Ca² activated Cl⁻ channel blocker disulfonic acid stilbene (DIDS, 300 μM) only inhibited part of the CCH-induced Isc response, while nonselective Cl⁻ channel blocker diphenylamine-dicarboxylic acid (DPC, 1 mM) reduced all, indicating the involvement of different conductance pathways. Both peaks of the CCH-induced Isc response could be significantly inhibited by pretreatment with an adenylate cyclase inhibitor, MDL12330A (50 μM). An increase in intracellular cAMP content upon stimulation of CCH was measured. All of the initial peak and part of the second peak could be inhibited by pretreatment with Ca² -chelating agent BAPTA/AM (50 μM) and an endoplasmic reticulum Ca² pump inhibitor, Thapsigagin (Tg, 1 μM). In a whole-cell patch clamp experiment, CCH induced an inward current in the single cell. Two different profiles of currents were found; the first component current exhibited an outward rectifying I-V relationship in a time and voltage-dependent manner, and the current followed showed a linear I-V relationship. The carbachol-induced current was found to be partially blockable by DIDS and could be completely blocked by DPC. The above results indicate that the CCH-induced Cl⁻ secretion could be mediated by Ca² and cAMP-dependent regulatory pathways.