The relaxant action of adenine nucleotides was studied in isolated rabbit trachealis to assess the presence of P2-purinoceptors in the airways, their cellular location, and pharmacologic properties. Strips of tracheal smooth muscle with intact epithelium were incubated in tissue baths and contracted with 1 microM acetylcholine. Over a dose range of 0.1 microM to 1 mM, ATP and ADP were significantly more potent than adenosine in relaxing tracheal smooth muscle. Significant relaxations were also elicited by AMP-PCP, AMP-CPP, and AMP-PNP, three ATP analogs stable to enzymatic hydrolysis to adenosine. In the absence of acetylcholine, neither ATP nor AMP-CPP exerted any contractile effect on the tracheal strips. In tissues selectively denuded of epithelium, ATP-, ADP-, and AMP-PCP-induced relaxations were markedly reduced. ATP-induced relaxation was also inhibited by the P2y-purinoceptor antagonist Reactive Blue 2 (RB2) (50 to 300 microM) and partially reduced by the cyclooxygenase inhibitor indomethacin (10 microM), whereas adenosine-induced relaxation was not significantly affected by these agents. These results suggest that ATP can induce smooth muscle relaxation in acetylcholine-contracted tracheal strips through a distinct P2-purinoceptor. This receptor appears to be located on the epithelium where its relaxant effect is mediated in part by release of one or more cyclooxygenase products. Additional relaxation at high ATP concentrations may occur through enzymatic hydrolysis of ATP to adenosine and interaction at P1-purinoceptors.