The patch-clamp technique was used to examine the tolbutamide-sensitivity of the adenosine 5'-triphosphate (ATP)-dependent K+ channel in mouse pancreatic B-cells. When studied at 37 degrees C in cell-attached membrane patches, this channel had a single-channel conductance of 88 pS and was half-maximally inhibited by 2.2 mumol/l tolbutamide in the presence of 3 mmol/l D-glucose and 10 mumol/l nifedipine. The tolbutamide-induced decrease in the amplitude of the single-channel currents indicated that the membrane potential was sufficiently depolarized for initiation of insulin release by 30 but not by 10 mumol/l of tolbutamide. Using 300 mumol/l diazoxide to open the ATP-dependent K+ channels already closed by 3 mmol/l D-glucose alone, it was demonstrated that initiation of insulin release requires closure of more than 98% of all ATP-dependent K+ channels. In excised inside-out membrane patches, the K+ channel-blocking potency of tolbutamide was maximally enhanced by 0.3 mmol/l adenosine 5'-diphosphate (ADP) at the cytoplasmic side. This ADP effect required the presence of Mg2+. Inhibition of K+ channel activity by ATP, ADP (Mg2(+)-free) or their non-hydrolyzable analogues adenylyl-imidodiphosphate (AMP-PNP) and alpha, beta methylene adenosine 5'-diphosphate (AMP-CP) was not accompanied by enhancement of tolbutamide-sensitivity. The results suggest that cytosolic MgADP controls tolbutamide-sensitivity by interaction with a receptor site not identical with the site mediating channel closure and that this control plays a role in the intact B-cell.