The enteric nervous system (ENS) controls gut function. P2X receptors and nicotinic acetylcholine receptors (nAChRs) are ligand-gated cation channels that mediate fast synaptic excitation in the ENS. Close molecular coupling in enteric neuronal membranes contributes to a mutually inhibitory interaction between these receptors; this effect is called cross-inhibition. We studied the molecular mechanisms responsible for cross-inhibition. Whole cell patch-clamp techniques were used to measure P2X- and nAChR-mediated currents in cultured enteric neurons and HEK-293 cells. In cultured myenteric neurons, ACh (3 mM) and ATP (1 mM) coapplication evoked an inward current that was only 57 +/- 6% (P < 0.05) of the predicted current that would have occurred if the two populations of channels were activated independently. In HEK-293 cells coexpressing alpha(3)beta(4) nAChR/P2X(2) receptors, coapplication of ATP and ACh caused a current that was 58 +/- 7% of the predicted current (P < 0.05). To test the importance of P2X subunit COOH-terminal tail length on cross-inhibition, P2X(3) and P2X(4) subunits, which have shorter COOH-terminal tails, were studied. Cross-inhibition with alpha(3)beta(4) nAChRs and P2X(3) or P2X(4) subunits was similar to that occurring with P2X(2) subunits. P2X receptor or alpha(3)beta(4) nAChR desensitization did not prevent receptor cross-inhibition. These data indicate that the alpha(3)beta(4)-P2X receptor interaction is not restricted to P2X(2) subunits. In addition, active and desensitized conformations of the P2X receptor inhibit nAChR function. These molecular interactions may modulate the function of synapses that use ATP and ACh as fast synaptic transmitters in the ENS.