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The present study investigated the role of pituitary adenylate cyclase-activating polypeptide (PACAP) in the regulation of electrogenic anion secretion by the rat cauda epididymal epithelium. PACAP38, which has been shown to affect secretory function in various exocrine and endocrine tissues, gave rise to a concentration-dependent increase in the short-circuit current (Isc). The PACAP38 effect was restricted to the apical aspect of the epididymal cells. The Isc response to PACAP38 was abolished in Cl-(-)free solution and completely inhibited by the Cl- channel blocker, diphenylamine-dicarboxylic acid. The Isc response to PACAP38 was also suppressed by pretreatment of the cells with the adenylate cyclase inhibitor, MDL12330A. The localization of PACAP38 was further investigated using an immunohistochemical technique. PACAP38 immunoreactivity was observed in the cauda epididymal tubules as well as in the cultured epithelium, indicating its epithelial origin. The present results suggest that Cl- secretion in the epididymis may be regulated by PACAP38, which could be locally synthesized and released by the epithelial cells, in a paracrine or autocrine fashion.