1. Genistein (GST), an inhibitor of protein tyrosine kinase (PTK), Na3VO4 (VO4), an inhibitor of phosphotyrosine phosphatase (PTPase), and forskolin (FSK), an activator of the cyclic AMP-dependent, cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel, were applied to guinea-pig ventricular myocytes to probe for a possible role of tyrosine phosphorylation in the regulation of cardiac Cl- channels. 2. Myocytes in the standard whole-cell configuration were pulsed to various potentials and Cl- current (ICl) measured as the difference from control background current. GST (1-500 microM) activated a current that had similar biophysical properties (time- and voltage-independent; Cl(-)-dependent reversal potential and outward rectification) as ICl activated by 5 microM FSK. The EC50 for activation of Cl- conductance (gCl) by GST was approximately 100 microM, and gCl activated by GST (500 microM) was as large as gCl activated by maximally-effective FSK (5 microM). Daidzein, a GST analogue with little effect on PTK, was at least one order less effective than GST. 3. GST responses were rapidly and reversibly inhibited by 0.1-1 mM VO4 treatments that had little effect on FSK-activated ICl. 4. Niflumic acid (100-200 microM) reversibly depressed GST (100 microM)-activated gCl by 55%. 5. GST (50 microM) strongly incremented current in myocytes with cyclic AMP-dependent CFTR ICl already activated by maximally-effective FSK 5 microM. 6. Based on these results, and on evidence of a synergistic interaction between GST and FSK, we conclude that inhibition of tyrosine phosphorylation by GST causes an activation of cardiac CFTR that is not mediated by an elevation of cyclic AMP.