K+ channels sensitive to intracellular ATP (KATP channels) have been described in a number of cell types and are selectively inhibited by sulfonylurea drugs. To look for the presence of this type of K+ channel in the basolateral membrane of tight epithelia, we have used an amphibian renal cell line, the A6 cells, grown on filters. After the selective permeabilization of the apical membrane with amphotericin B, the basolateral conductance was studied under voltage-clamp conditions. Tolbutamide inhibited 65.8 +/- 6.3% of the barium-sensitive current. The tolbutamide-sensitive conductance had an equilibrium potential of -83 +/- 1 mV and was inward rectifying in spite of the outwardly directed K+ gradient. Similar results were obtained with glibenclamide. The half-inhibition constants were 25.7 +/- 3.0 microM and 0.114 +/- 0.018 microM for tolbutamide and glibenclamide, respectively. To study the relation between cellular ATP and the activity of this conductance, A6 cells were treated with glucose (5 mM) and insulin (250 microU/ml). This maneuver significantly increased the cellular ATP and abolished the tolbutamide-sensitive conductance. A sulfonylurea-sensitive K+ conductance is present and active in the basolateral membrane of A6 cells. This conductance appears to be modulated by physiological changes of intracellular ATP.