Because some endothelial cells contain a high density of functional vasoactive intestinal peptide (VIP) receptors, it is possible that in some cases, relaxation of blood vessels by VIP is mediated by endothelium. We showed earlier that VIP inhibited inwardly rectifying K+ currents (IKin) in cultured bovine pulmonary artery endothelial cells. Our studies now provide both direct and indirect evidence that activation of these receptors does not occur through an elevation of cAMP level in these cells. Isoproterenol increased cAMP in endothelial cells from 30% to 35% over the basal levels. In contrast, VIP did not elevate cAMP in endothelial cells and even decreased it in some instances. In whole-cell patch-clamp experiments, isoproterenol weakly inhibited the IKin (about 80% less than VIP). The magnitudes of effects evoked by other activators of the cAMP cascade (forskolin, cAMP analogs) on this current were intermediate between those of VIP and isoproterenol. Although cAMP elevation can reduce the IKin current in endothelial cells, it is not responsible for the inhibitory effect of VIP on this current. We demonstrated that VIP receptors interact with the IKin channels through a G protein. Guanosine 5'-(3'-O-thiotriphosphate, a nonhydrolyzable GTP analog, or cholera toxin inhibited these channels in a manner similar to inhibition by VIP. The activity of the IKin channels was pertussis toxin-insensitive. Furthermore, guanosine-5'-O-(2-thiodiphosphate) blocked the VIP receptor-mediated effect on the IKin. Our results suggest that VIP receptors couple to IKin channels through a G protein.