The neuropeptide bombesin acts on a variety of target cells to stimulate the processes of secretion and cell proliferation. In this study we determined whether bombesin receptors interact with known guanine nucleotide-binding proteins in four different cell types: GH4C1 pituitary cells, HIT pancreatic islet cells, Swiss 3T3 fibroblasts, and rat brain tissue. Maximal concentrations of nonhydrolyzable GTP analogs decreased agonist binding to bombesin receptors in membranes from all four sources. In GH4C1 and HIT cell membranes GTP analogs inhibited bombesin receptor binding with IC50 values of about 0.1 microM, whereas GDP analogs were approximately 10-fold less potent. In contrast, GMP and the nonhydrolyzable ATP analog adenylyl-imidodiphosphate had no effect at 100 microM. Equilibrium binding experiments in GH4C1 and HIT cell membranes indicated a single class of binding sites with a dissociation constant (Kd) for [125I-Tyr4]bombesin of 24.4 +/- 7.0 pM and a binding capacity of 176 +/- 15 fmol/mg protein. Guanine nucleotides decreased the apparent affinity of the receptors without significantly changing receptor number. Consistent with this observation, guanine nucleotides also increased the rate of ligand dissociation. Pretreatment of GH4C1 or HIT cells with either pertussis toxin (100 ng/ml) or cholera toxin (500 ng/ml) for 18 h did not affect agonist binding to membrane bombesin receptors, its regulation by guanine nucleotides, or bombesin stimulation of hormone release. Although pertussis toxin pretreatment has been reported to block bombesin stimulation of DNA synthesis in Swiss 3T3 cells, it did not alter the binding properties of bombesin receptors in Swiss 3T3 membranes or inhibit the rapid increase in intracellular [Ca2+] produced by bombesin in these cells. In summary, our results indicate that the bombesin receptor interacts with a guanine nucleotide-binding protein which exhibits a different toxin sensitivity from those which regulate adenylate cyclase as well as those which couple some receptors to phospholipases.