We have examined the cultured human bronchial epithelial cells (16HBE) to learn if changes in Cl(-) concentration or osmolality stimulate the cells to release ATP and to determine whether its release is cyclic AMP (cAMP)- and/or Ca(2+)-dependent by using the luciferin-luciferase luminometric assay. In a control solution (290 mosmol kg H(2)O(-1)), the external ATP concentration and the rate of ATP release were 0.52 +/- 0.20 nM and 0.036 +/- 0.034 pmol min(-1), respectively. Upon hypotonicity (205 mosmol kg H(2)O(-1)), they increased to 7.0 +/- 1.3 nM and 3.1 +/- 0.6 pmol min(-1), respectively, at 6 min, then decreased. At the peak, the rate of ATP release is estimated to be 6.2x10(4) ATP molecules s(-1) per cell. An accumulation of the released ATP for the initial 10 min increased significantly (p < 0.005) by 71.5% in the presence of forskolin (10 microM), adenylyl cyclase activator, however, it was abolished (p < 0.001) by pretreatment with BAPTA-AM (25 microM), a membrane permeable Ca(2+) chelator. On the other hand, neither low Cl(2-) (75 mM, isotonic) nor hypertonicity (+NaCl or +mannitol, 500 mosmol kg H(2)O(-1)) could significantly increase the ATP release. Further, forskolin or ionomycin (a Ca(2+) ionophore) or, both, failed to stimulate ATP release under the isotonic condition. In conclusion, first, hypertonicity and changes in Cl(-) concentrations are not effective signals for the ATP release; second, hypotonicity-induced ATP release is potentiated by the level of intracellular Ca(2+) and cAMP; and third, a biphasic increase in ATP release and its low rate at the peak support the hypothesis that ATP is released through a non-conducting pathway model, such as exocytosis, or through a volume-dependent, ATP-conductive anion channel.