Extracellular ATP can stimulate mucin release from primary hamster tracheal surface epithelial (HTSE) cells via a P2 purinoceptor-mediated mechanism, based on agonist potency studies of mucin release (Br. J. Pharmacol. 1991; 103:1053-1056). In the present study, we examined the kinetics of ATP binding to the surface of intact HTSE cells at 4 degrees C using ATP gamma S35 as a radioligand. We found that ATP gamma S35 bound to HTSE cells in a saturable, reversible manner, reaching an equilibrium at about 30 min. Scatchard analysis of equilibrium binding suggested the presence of two binding sites with Kd values of 0.47 and 9.4 microM. Competitive binding experiments, based on the ability of nucleotides and ATP analogs to block ATP gamma S35 revealed a rank order of ATP > ADP > alpha,beta-methylene ATP > 2-methylthio ATP > or = beta, gamma-methylene ATP. Neither AMP nor adenosine could inhibit the ATP gamma S35 binding. A comparison between the ability of nucleotides to compete with ATP gamma S35 binding and their ability to induce mucin release revealed a rather poor correlation (r2 = 0.67) with all of the above nucleotides but a good correlation (r2 = 0.96) without 2-methylthio ATP, indicating the presence of heterogenous ATP binding sites on the HTSE cell surface. UTP, a pyrimidine nucleotide, which is almost equipotent with ATP in its ability to stimulate mucin release, was much less potent than ATP in its ability to displace the ATP gamma S35 binding in these HTSE cells.