α1-Adrenergic receptor stimulation can inhibit the Cl- current activated by β-adrenergic receptor agonists in guinea-pig ventricular myocytes. We investigated the role of G proteins in mediating this type of α-adrenergic response. The combined α-and β-adrenergic agonist norepinephrine (NE) activated the Cl- current with an EC50 value of 53 nM. Preincubation of myocytes with PTX decreased the EC50 value for NE activation of the Cl- current to 5.9 nM, and addition of the α1-adrenergic receptor antagonist prazosin did not cause any further change in sensitivity to NE. These results suggest that the α1-adrenergic inhibition of β- adrenergic responses is mediated through a PTX-sensitive G protein. However, PTX pretreatment also increased the sensitivity of the Cl- current to the selective β-adrenergic agonist isoproterenol (Iso), which indicates that the PTX treatment increases the sensitivity to β-adrenergic stimulation alone and that this could account for the PTX-induced change in sensitivity to NE. Consistent with this idea, the selective α1-adrenergic receptor agonist methoxamine was still able to inhibit the Cl- current activated by Iso in PTX-treated myocytes. However, the sensitivity to methoxamine was significantly decreased. In control cells, the Cl- current activated by 30 nM Iso was inhibited by methoxamine with an EC50 value of 8.3 μM, but in PTX-treated cells, the EC50 value was 284 μM. The EC50 for methoxamine inhibition was similarly increased when the Cl- current was activated by 300 nM Iso. These data suggest that the effects of PTX on α1-adrenergic responses can actually be explained by changes in the sensitivity to β- adrenergic stimulation. To verify the role for a G protein in mediating the inhibitory α1-adrenergic response, we examined the effect of methoxamine on the Cl- current activated in cells dialyzed with the nonhydrolyzable GTP analogue guanosine-5'-O-(3-thio)-triphosphate. Pre-exposure to methoxamine resulted in an attenuated response upon subsequent exposure to Iso alone. We conclude that α1-adrenergic inhibition of β-adrenergic responses is mediated by a G protein-dependent mechanism that appears to be PTX- insensitive.