The actions of microelectrophoretically administered substance P on Renshaw cells in pentobarbitone anesthetized cats were investigated. The effects on spontaneous and synaptic firing and interactions with a number of other agents including acetylcholine, acetyl-.beta.-methylcholine, acidic amino acids, morphine, dihydro-.beta.-erythroidine and strychnine were studied to elucidate the mechanism of action of substance P. Substance P usually selectively depressed the excitation by ACh [acetylcholine], and also reduced submaximal synaptically evoked discharges which activate nicotinic receptors, but failed to modify excitation caused either by acetyl-.beta.-methylcholine, which activates muscarinic receptors, or excitation caused by glutamate or homocysteate. Substance P also depressed excitation by morphine which acted via the nicotinic receptors. The inhibitory effect was not blocked by strychnine and was considered unlikely to be due to interaction between the polypeptide and either glycine or GABA receptors. On some cells substance P caused excitation which was blocked by dihydro-.beta.-erythroidine. Mixed excitatory-inhibitory effects were observed on some of these neurons. Substance P could function as a synaptic inhibitory mediator with an unusual selectivity of action.