We used the short-circuit current (I_sc) technique to investigate the effects of the isoflavone, genistein, on the electrogenic Cl^- secretion of the mouse jejunum. Genistein stimulated a sustained increase in I_sc that was dose dependent. Bumetanide inhibited 76 ± 5% of the genistein-stimulated I_sc consistent with activation of Cl^- secretion. Genistein failed to stimulate I_sc following maximal activation of the cAMP pathway by forskolin. Additionally, forskolin had a reduced effect on I_sc of the mouse jejunum in the presence of genistein. Glibenclamide, a blocker of CFTR, eliminated the genistein-stimulated increase of I_sc and reduced the forskolin-stimulated I_sc. Clotrimazole, a Ca²⁺-activated K⁺ channel blocker, failed to reduce the genistein-stimulated I_sc. Vanadate, a blocker of tyrosine-dependent phosphatases, reduced the genistein-activated I_sc. Tryphostin A23, a tyrosine kinase inhibitor, reduced basal I_sc, afterwhich, genistein failed to stimulated I_sc. These data suggest that genistein activated a sustained Cl^- secretory response of the mouse jejunum and that the effect of genistein is via a tyrosine-dependent phosphorylation pathway.