P2X receptors are ATP-gated cation channels composed of one or more of seven different subunits. P2X receptors participate in intestinal neurotransmission but the subunit composition of enteric P2X receptors is unknown. In this study, we used tissues from P2X3 wild-type (P2X3+/+) mice and mice in which the P2X3 subunit gene had been deleted (P2X3-/-) to investigate the role of this subunit in neurotransmission in the intestine. RT-PCR analysis of mRNA from intestinal tissues verified P2X3 gene deletion. Intracellular electrophysiological methods were used to record synaptic and drug-induced responses from myenteric neurons in vitro. Drug-induced longitudinal muscle contractions were studied in vitro. Intraluminal pressure-induced reflex contractions (peristalsis) of ileal segments were studied in vitro using a modified Trendelenburg preparation. Gastrointestinal transit was measured as the progression in 30 min of a liquid radioactive marker administered by gavage to fasted mice. Fast excitatory postsynaptic potentials recorded from S neurons (motoneurons and interneurons) were similar in tissues from P2X3+/+ and P2X3-/- mice. S neurons from P2X3+/+ and P2X3-/- mice were depolarized by application of ATP but not alpha,beta-methylene ATP, an agonist of P2X3 subunit-containing receptors. ATP and alpha,beta-methylene ATP induced depolarization of AH (sensory) neurons from P2X3+/+ mice. ATP, but not alpha,beta-methylene ATP, caused depolarization of AH neurons from P2X3-/- mice. Peristalsis was inhibited in ileal segments from P2X3-/- mice but longitudinal muscle contractions caused by nicotine and bethanechol were similar in segments from P2X3+/+ and P2X3-/- mice. Gastrointestinal transit was similar in P2X3+/+ and P2X3-/- mice. It is concluded that P2X3 subunit-containing receptors participate in neural pathways underlying peristalsis in the mouse intestine in vitro. P2X3 subunits are localized to AH (sensory) but not S neurons. P2X3 receptors may contribute to detection of distention or intraluminal pressure increases and initiation of reflex contractions.