Motility of gastrointestinal smooth muscle is effectively modified by non-cholinergic non-adrenergic neurotransmission. Here, we studied the impact of purinergic signaling on gut motility. To this end, smooth muscle stripes of the rat isolated ileum were fixed in an organ bath in order to record isometric contractions and relaxations. The P2 purinoceptor agonist ATP transiently induced a relaxation that was followed by a long-lasting contraction of the ileal smooth muscle stripes. This biphasic response was also obtained in experiments using the more specific P2X receptor agonist a,b-methylene-ATP. Pre-incubation with the P2 receptor antagonist suramin significantly reduced the ATP-induced contraction, but left the ATP-induced relaxation unaltered. In contrast, both relaxations and contractions induced by a,b-methylene-ATP were significantly reduced in smooth muscle stripes pre-treated with suramin. These results indicate that purinergic signaling leads to a biphasic change in ileal motility consisting of a transient relaxation followed by a sustained contraction both of which are mediated by P2X receptor activation. In addition, ATP may also cause transient relaxations by further mechanisms which are independent of P2X receptors.