The nature of the opiate modulation of adenylate cyclase following acute and chronic agonist exposure has been investigated in rat spinal cord. Using membranes of both adult rat spinal cord and spinal cord-dorsal root ganglion cocultures, we found that kappa-opiate receptors are negatively coupled to adenylate cyclase. The kappa-opiate agonists (e.g., U50488) inhibit significantly and dose-dependently the basal and the forskolin-stimulated cyclase activities, whereas mu and delta agonists are ineffective. The regulatory action is stereospecific and requires the presence of GTP. EGTA treatment of the plasma membranes abolished the effect of kappa-opiate agonists on the basal cyclase activity, and this inhibitory effect could not be restored by subsequent addition of Ca2+. The EGTA treatment did not affect the kappa agonist inhibition of the forskolin-stimulated cyclase. The results also show that following chronic exposure of cultured cells to etorphine or U50488, there is a loss of kappa agonist inhibition of the cyclase. Moreover, this desensitization process appears to be heterologous, because alpha 2-adrenergic agonists (e.g., clonidine or norepinephrine) and the muscarinic agonist (carbachol) exhibited significantly lower potency for inhibiting cyclase activity when compared to untreated cultures. This pattern of heterologous desensitization suggests that chronic exposure to kappa opiates leads to alterations in postreceptor regulatory components, possibly GTP-binding proteins.