There is growing evidence that secretin, the first hormone discovered in our history, has functions in the brain other than in the gastrointestinal tract. This article reports for the first time that secretin and its receptor mRNAs are produced in distinct cell types within the epididymis. To test if secretin affects electrolyte transport in the epididymis, we measured short-circuit current (Isc) in cultured epididymal epithelia and found secretin dose-dependently stimulated Isc. Ion substitution experiments and use of pharmacological agents inferred that the stimulated Isc is a result of concurrent electrogenic chloride and bicarbonate secretion. It is further shown that secretin and pituitary adenylate cyclase-activating polypeptide (PACAP) function via totally different mechanisms: 1) PACAP works only from the apical side of the epithelium to stimulate chloride and not bicarbonate secretion, while secretin acts on the apical and basolateral sides to stimulate chloride and bicarbonate secretion. 2) the stimulation by PACAP but not secretin requires local prostaglandin synthesis. By immunocytochemical staining, secretin is localized in the principal cells of the initial segment and caput epididymidis, whereas secretin receptor is present in the principal cells of the proximal as well as the distal part of the epididymis. This pattern of distribution appears to be consistent with the idea that secretin is secreted by the proximal epididymis and acts on the proximal and distal epididymis in an autocrine and paracrine fashion. Its function is to control secretion of electrolytes and water.