Bombesin- and vasopressin-stimulated phospholipase D (PLD) activities are rapidly desensitized in 3T3 cells, in addition both agonists are subject to heterologous desensitization. Binding studies showed that homologous desensitization was partly a result of loss of cell surface receptors, whilst heterologous desensitization was independent of receptor changes. Pretreatment with either agonist reduced subsequent GTP gamma S-stimulated PLD activity by 50% whereas a pretreatment with GTP gamma S did not attenuate the response, suggesting that the G-protein or downstream effector systems were affected by receptor activation resulting in desensitization. The desensitization of receptor-stimulated PLD activation provides support for the phospholipase functioning in a key signalling pathway.