In higher concentrations (> 3 x 10(-4) M) than those causing contractile responses. ATP relaxed the smooth muscle of the guinea pig perfused trachea. Here we examined the relaxant effects of nucleotides. ATP and its nonhydrolyzable congener, beta, gamma-methylene ATP (APPCP), were approximately 4- and approximately 117-fold, respectively, more potent when applied separately to the serosal (extraluminal, EL) surface compared to the mucosal (intraluminal, IL) surface of methacholine (3 x 10(-7) M; EL)-contracted tracheae. APPCP was orders of magnitude more potent than ATP in both EL and IL compartments. EL UTP did not cause relaxation; IL UTP was nearly devoid of activity. The order of EL and IL activity (APPCP > ATP) was unusual for nucleotide-induced relaxation of smooth muscle. Relaxation to ATP was not inhibited by the Cl- channel blocker 4,4'-diisothiocyano-2,2'-stilbene disulfonate (10(-4) M) or by the cyclo-oxygenase inhibitor indomethacin (3 x 10(-6) M), in contrast to the inhibitory effects of these drugs on contraction to ATP. The adenosine receptor antagonist 8-phenyltheophylline (10(-6) M) had no effect on relaxation to ATP or APPCP. Our findings indicate that Cl- channels, prostaglandins and adenosine are not involved in relaxation to adenine nucleotides.