The effects of intracellular purine nucleotides on tolbutamide-induced block of ATP-dependent K+ channels from mouse pancreatic B-cells were studied using the patch-clamp technique. When applied to the inside of excised patches, tolbutamide alone blocked channel activity half-maximally at 55 microM and the concentration-response curve for the inhibition of K+ channels by tolbutamide was flat. ADP (1 mM), but not other nucleotides (AMP, GTP or GDP) increased the steepness of the concentration-response curve and decreased the half-maximally effective tolbutamide concentration to 4.2 microM. It is suggested that the ATP-dependent K+ channel or a closely related structure contains a receptor which is accessible for cytosolic ADP and controls the sensitivity to tolbutamide.