The effect of genistein on anion secretion via cystic fibrosis transmembrane conductance regulator (CFTR) in cultured rat cauda epididymal epithelia was studied by short-circuit current (Isc) technique. Genistein added apically stimulated a concentration-dependent rise in Isc due to Cl⁻ and HCO₃⁻ secretion. The genistein-induced Isc was observed in basolaterally permeabilized monolayers, suggesting that the Isc response was mediated by the apical anion channel. The response could be blocked by the nonspecific Cl⁻ channel blocker, diphenylamine-2-carboxylate (DPC), but not by the Ca² -activated Cl⁻ channel blocker, 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS). Genistein did not increase intracellular cAMP, but H-89, a protein kinase A inhibitor, completely abolished the Isc response to genistein. Moreover, pretreatment of the tissues with MDL-12330A, an adenylate cyclase inhibitor, markedly attenuated the Isc response to genistein, but the response was restored upon the addition of exogenous cAMP. Ca² , protein kinase C, tyrosine kinase, and protein phosphatase signalling pathways were not involved in the action of genistein. It is speculated that genistein stimulates anion secretion by direct interaction with CFTR. This requires a low level of phosphorylation of CFTR by basal protein kinase A activity. It is suggested that genistein may provide therapeutic benefit to male infertility associated with cystic fibrosis.