There is a well-characterized membrane chloride current (ICl, cAMP) in the heart that can be activated by β-adrenergic agonists and is due to expression of the cardiac isoform of the epithelial cystic fibrosis transmembrane conductance regulator (CFTR). We have investigated whether 17β-estradiol (E2) modulates ICl, cAMP in single ventricular myocytes. Under whole-cell tight-seal voltage-clamp conditions, ICl, cAMP was evoked by exposing cells to 20 nM isoprenaline. On the addition of 30 µM E2, membrane slope conductance, measured at potentials near 0 mV, increased over that induced by isoprenaline alone by 2.46±0.16 (p< 0.001). The effects of E2 were concentration-dependent and described by a Hill Plot with an EC50 of 8.2 µM and a Hill coefficient of 1.63. The application of membrane-impermeant E2 conjugated to bovine serum albumin (E2-BSA) potentiated isoprenaline-evoked ICl, cAMP by approximately the same degree as that for the equivalent level of free E2. Cell surface binding was observed with confocal microscopy by using BSAFITC tagged E2. This binding was inhibited by nonlabeled, nonconjugate E2, the specific E2 antagonist ICI 182,780, and incubation of E2coBSA with a specific anti-E2 antibody (E2885). ICI 182,780 (100 µM) significantly reduced the increase in ICl, cAMP evoked by 10 µM E2 to 1.46±0.10 (p< 0.02). The preincubation of myocytes with the NOS inhibitor N-ω-nitro-arginine (L-NNA, 1 mM) reduced the potentiation of ICl, cAMP by 30 µM E2, to 1.93±0.06 (p< 0.02), and for 10 µM E2, to 1.32±0.05 (p< 0.002). E2 also increased ICl, cAMP evoked by bath application of 0.5 µM Forskolin. These experiments demonstrate that, under our experimental conditions, E2 dramatically increases ICl, cAMP in ventricular myocytes by mechanisms involving a contribution by NOS, but that can be only partially accounted for through binding to classical plasma membrane estrogen receptor sites. This potentiation of ICl, cAMP by E2 may play a significant role in the observed clinical actions of E2 on the incidence of cardiac arrhythmias and hypertrophy.