Novel insulinotropic agent nateglinide stimulates insulin via binding to sulfonylurea receptor and closing the ATP-dependent K + (K A T P ) channels in pancreatic β-cells, leading to an increase in [Ca 2 + ] i for exocytosis. The voltage-dependent Ca 2 + channel and the delayed rectifier K + (Kv) channels are also present in β-cells and their activities determine the configuration of action potential and hence contribute to the regulation of [Ca 2 + ] i and insulin secretion. This study, by using the patch-clamp method in whole cell configuration, comparatively characterized the direct effects of sulfonylurea receptor ligands including nateglinide, glyburide, and repaglinide on Kv and Ca 2 + channels. Each agent inhibited Kv currents in a concentration-dependent manner with effective concentration range two to three orders higher than that for blocking K A T P channels. A marginal stimulation of Ca 2 + current was observed with all drugs, while repaglinide at concentration greater than 300 nM inhibited Ca 2 + current. The direct effects of these antidiabetic agents on Kv and Ca 2 + channels may act concertedly with their primary action on K A T P channels in regulating [Ca 2 + ] i and the stimulus-secretion coupling.