Cultures epithelial monolayers of MDCK cells were grown upon Millipore filter supports and mounted in Ussing chambers for ion-transport studies. Addition of exogenous ATP to the basal bathing solutions resulted in a stimulation of the short-circuit current which was due to both an increased transmonolayer p.d. and an increased conductance. Measurements of tracer Na+ and Cl- fluxes demonstrate that the ATP-stimulated short-circuit current, results from basal to apical Cl- movement (secretion) across the cultured monolayer. ATP-stimulated net Cl- secretion was inhibited by furosemide (1 x 10(-4) M) added to the basal bathing solution and by elevating the basal medium K+ concentration from 5.4 to 54 mM. Both furosemide and elevated basal K+ exert their inhibitory action upon the ATP-dependent short circuit current primarily by abolishing the electrogenic component without affecting the increased transmonolayer conductance. Hyperpolarization of the transmonolayer potential difference by applied currents also reduces the ATP dependent increase in the short-circuit current. The increased short-circuit current was insensitive to replacement of medium Na+ by choline+, but was linearly related to Cl- concentration with isethionate (2-hydroxyethanesulphonate) replacements. NO3-, I-, and the thiocyanate anion were all ineffective substitutes for Cl- whereas Br- and acetate were only partially effective. Sodium thiocyanate (10 mM) in the presence of NaCl inhibited the ATP-stimulated short-circuit current.