The colocalization of acetylcholine (ACh) and neuropeptides (e.g., substance P and enkephalins) in the splanchnic nerve terminals suggests that these compounds might interact to modulate adrenal catecholamine release. Use has been made of primary monolayer and suspension cultures of bovine adrenal chromaffin cells to investigate postsynaptic receptor interactions between acetylcholine and a number of neuropeptides endogenous to the adrenal medulla and splanchnic nerve. The cells have both nicotinic and muscarinic acetylcholine receptors, but only the nicotinic receptors stimulate catecholamine release. Substance P, somatostatin, and the enkephalins all produced an inhibition of the Ach-evoked secretion of catecholamines, but their potency ranged over 100-fold. Substance P was the most potent with a mean inhibitory concentration (IC50) of 10−6 M and Leu-enkephalin the least potent with an IC50 > 10−4 M. These pharmacological effects were monitored conveniently by measuring the release of [3H] norepinephrine preloaded into the cells or alternatively, "on-line" by measuring ATP released into an incubation medium containing luciferin and firefly tail extract (luciferase). Of interest, the endogenous enkephalin heptapeptide (Met-enkephalin Arg6-Phe7) and "big" Met-enkephalin (BAM-22P) were some 100-fold more effective than Leu- or Met-enkephalin at inhibiting the nicotinic secretion of catecholamines, suggesting that a unique opiate receptor may be involved. Substance P had two distinct actions on the nicotinic response: (1) substance P inhibited acetylcholine-induced release of catecholamines; and (2) substance P protected against acetylcholine-induced desensitization of catecholamine release. With regard to (1), substance P inhibited the secretion of catecholamines and ATP evoked by acetylcholine or nicotine but not that evoked by K+ or veratridine, nor did substance P by itself affect secretion. Substance P appeared to interact with a regulatory site on the acetylcholine receptor – ionophore complex. Substance P receptors on chromaffin cells have similar structural requirements for activation as do substance P receptors in other substance P responsive tissues. With regard to (2), substance P (> 5 × 10−6 M) completely protected against desensitization of catecholamine release produced by acetylcholine (> 10−4 M) or nicotine (> 2.5 × 10−6 M) with no effect on K+-induced desensitization. Substance P protected against both a Ca2+-dependent and a Ca2+-independent component of desensitization and this protection against desensitization was a result of facilitation of catecholamine release rather than inhibition of catecholamine reuptake. These actions of substance P may help to ensure a prolonged adrenal catecholamine output during stress.