Substance P is known to inhibit nicotinic acetylcholine receptors from neuronal tissue, skeletal muscle, and electroplaque. The interaction of substance P with specific combinations of neuronal nicotinic acetylcholine receptor subunits was studied by expressing various combinations of subunits in Xenopus oocytes. The response to acetylcholine was inhibited by substance P with all subunit combinations tested; however, the apparent affinity for substance P varied by 20-30-fold. The affinity seemed to be dependent on the beta subtype expressed (beta 4 or beta 2). This suggests that the beta subunit may contribute, at least partially, to the substance P binding site. In the case of the alpha 7 subtype, which forms a homooligomeric receptor, the apparent affinity for substance P was intermediate between those of the two beta subtypes coexpressed with either alpha 3 or alpha 4. As previously found, the inhibition was noncompetitive. Furthermore, the inhibition was not voltage dependent and, therefore, is unlikely to be due to substance P blocking the channel within the transmembrane portion of the pore.