Previously, we demonstrated that genistein stimulated Cl(-) secretion in the mouse jejunum (Baker MJ and Hamilton KL, Am J Physiol Cell Physiol 287: C1636-C1645, 2004); however, the mode of action of genistein still remains unclear. Here, we examined the activation of Cl(-) secretion by the modulation of phosphodiesterases (PDEs) by genistein (75 microM) in the mouse jejunum with the Ussing short-circuit current (I(sc)) technique. Drugs tested included theophylline (10 mM), a nonspecific PDE inhibitor; 8-methoxymethyl-3-isobutyl-1-methylxanthine (8-MM-IBMX; 100 microM), erythro-9-(2-hydroxyl-3-nonyl)-adenine (EHNA; 40 microM), milrinone (100 microM), and rolipram (40 and 100 microM), which are specific inhibitors of PDE1-PDE4, respectively. Theophylline stimulated a bumetanide-sensitive I(sc), indicative of Cl(-) secretion, and abolished genistein's stimulatory action on I(sc). Neither 8-MM-IBMX nor EHNA altered the basal I(sc) nor did these PDE inhibitors affect the stimulatory action of genistein on the I(sc) of the mouse jejunum. Rolipram had no effect on basal I(sc), but it reduced the genistein-stimulated I(sc) compared with time-matched control tissues. Milrinone stimulated a concentration-dependent increase in I(sc). Bumetanide (10 microM) inhibited 60 +/- 4% of milrinone-induced I(sc). Pretreating tissues with milrinone prevented genistein from stimulating I(sc), and pretreatment with genistein reduced the effect of milrinone on I(sc). H89 (50 microM), a PKA inhibitor, reduced the milrinone-stimulated I(sc). Likewise, H89 reduced the genistein-stimulated I(sc). Here, we demonstrate, for the first time, that genistein activates Cl(-) secretion of the mouse jejunum via inhibition of a PDE3-dependent pathway.