AbstractBackground  P2X2 and nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic excitation in the enteric nervous system. P2X receptors and nAChRs are functionally linked. This study examined the mechanisms responsible for interactions between P2X2 and α3β4subunit‐containing nAChRs.Methods  The function of P2X2 and α3β4 nAChRs expressed by HEK‐293 cells and guinea pig ileum myenteric neurons in culture was studied using whole‐cell patch clamp techniques.Key Results  In HEK‐293 cells expressing α3β4 nAChRs and P2X2 receptors, co‐application of ATP and acetylcholine caused inward currents that were 56 ± 7% of the current that should occur if these channels functioned independently (P < 0.05, n = 9); we call this interaction cross‐inhibition. Cross‐inhibition did not occur in HEK‐293 cells expressing α3β4 nAChRs and a C‐terminal tail truncated P2X2 receptor (P2X2TR) (P > 0.05, n = 8). Intracellular application of the C‐terminal tail of the P2X2 receptor blocked nAChR‐P2X receptor cross‐inhibition in HEK‐293 cells and myenteric neurons. In the absence of ATP, P2X2 receptors constitutively inhibited nAChR currents in HEK‐293 cells expressing both receptors. Constitutive inhibition did not occur in HEK‐293 cells expressing α3β4 nAChRs transfected with P2X2TR. Currents caused by low (≤30 μmol L−1), but not high (≥100 μmol L−1) concentrations of ATP in cells expressing P2X2 receptors were inhibited by co‐expression with α3β4 nAChRs.Conclusions & Inferences  The C‐terminal tail of P2X2 receptors mediates cross‐inhibition between α3β4 nAChR‐P2X2 receptors. The closed state of P2X2 receptors and nAChRs can also cause cross‐inhibition. These interactions may modulate transmission at enteric synapses that use ATP and acetylcholine as co‐transmitters.