The regulation by ATP of Cl- secretion in T84 cells grown on filters was investigated by measuring short-circuit current (Isc = net Cl- secretion). ATP (greater than or equal to 10 microM) added to the basolateral side markedly stimulated Isc both in the presence and absence of forskolin-activated Isc. Fluorescence microscopy of cells loaded with the Ca2+ indicator fura-2 showed that ATP stimulated a transient increase in intracellular free Ca2+ concentration [Ca2+]i. The augmentation of forskolin-stimulated Isc by ATP was at least partly caused by mobilization of Ca2+ from an internal store because prior depletion of the store using ionomycin prevented the response. The activity sequence for stimulation of Isc in the presence of forskolin was adenosine 5'-O-(3-thiotriphosphate) = 5'-adenylylimidodiphosphate (AMP-PNP) greater than ATP greater than ADP greater than AMP, suggesting the presence of a P2 purinergic receptor. Neither beta, gamma-methyleneadenosine 5'-triphosphate nor alpha, beta-methyleneadenosine 5'-triphosphate increased the Isc. Stimulation of Isc by ATP in the absence of forskolin was at least partly due to the breakdown of ATP to AMP and adenosine, which act at P1 receptors to stimulate Isc, since 1) inhibition of the ecto-phosphohydrolase 5'-nucleotidase by alpha, beta-methylene-ADP partially inhibited stimulation of Isc by ATP, 2) the adenosine receptor antagonists caffeine and 8-phenyltheophylline markedly inhibited the ATP-stimulated Isc, and 3) AMP-PNP, a weakly hydrolyzable analogue of ATP, caused a much smaller increase in Isc compared with ATP. Adenosine had no effect on [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS)