Prolonged exposure of Swiss 3T3 cells to vasopressin causes heterologous mitogenic desensitization to bombesin and structurally related peptides including gastrin-releasing peptide (GRP) without down-regulation of the bombesin receptor. The number and affinity of bombesin/GRP receptor sites and modulation of 125I-GRP binding by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) are unaffected in membrane preparations from vasopressin-treated cultures. Stimulation of inositol phosphate accumulation, mobilization of intracellular calcium, production of diacylglycerol, and transmodulation of the epidermal growth factor receptor by bombesin are similarly unaffected. Thus, the heterologous mitogenic desensitization is not due to uncoupling of bombesin receptor from transducing G protein(s) or to an inability to activate phospholipase C. Bombesin, unlike vasopressin, causes a rapid dose-dependent release of [3H]arachidonic acid and prostaglandin E2 from Swiss 3T3 cells (EC50 approximately 4 nM), which is inhibited by the specific bombesin receptor antagonist [Leu13-psi(CH2NH)-Leu14]bombesin. Crucially, release of [3H]arachidonic acid and prostaglandin E2 by bombesin is completely suppressed by prolonged pretreatment with vasopressin (EC50 = 0.6 nM). The mitogenic action of bombesin is restored by adding arachidonic acid to vasopressin-treated cells. We conclude first that arachidonic acid release is an early signal in the mitogenic response to bombesin and second that pretreatment with vasopressin induces heterologous mitogenic desensitization to bombesin by a novel mechanism: inhibition of arachidonic acid release.