The pancreatic K(ATP) channel, SUR1/Kir6.2, couples insulin secretion to the plasma glucose level. The channel is an octamer with four Kir6.2 subunits forming the pore and four sulphonylurea receptors (SUR1) regulating channel activity. SUR1 is an ABC protein with adenosine triphosphate (ATP)ase activity which activates the channel. It also contains the binding site for antidiabetic drugs like glibenclamide and repaglinide which close the channel by disrupting the stimulatory effect SUR-ATPase (MgATP-dependent) and by stabilising a long-lived closed channel state (MgATP-independent). In this study, we examined the effects of progressive truncation of the Kir6.2 N-terminus up to 20 amino acids on equilibrium binding and channel closure by glibenclamide and repaglinide, on the channel activating effect of the opener, 6-chloro-3-(1-methylcyclobutyl)amino-4H-thieno[3,2-e]-1,2,4thiadiazine 1,1-dioxide (NNC 55-0462), and on the binding kinetics of [(3)H]glibenclamide. Kir and SUR were transiently coexpressed in HEK cells and [(3)H]glibenclamide binding and patch-clamp experiments were performed in whole cells at 37°C and in isolated inside/out patches at 22°C. Truncation of the first 5 N-terminal amino acids abolished most of the affinity increase for glibenclamide and repaglinide that is produced by the association of Kir6.2 with SUR1. Progressive truncation continuously reduced the potency and efficacy of these drugs in closing the channel and impaired the ability to stabilise the closed state more than the ability to disrupt channel stimulation by SUR-ATPase. The effects of NNC 55-0462 were unchanged. Progressive truncation also speeded up dissociation of [(3)H]glibenclamide from the channel when dissociation was induced by an excess of (unlabelled) glibenclamide. This suggests the existence of a putative low affinity glibenclamide site on the channel whose affinity increases upon truncation. The data show that progressive truncation of the Kir6.2 N-terminus impairs the transduction of drug binding into channel closure more strongly than drug binding but leaves the effect of the opener NNC 55-0462 unchanged.