Antimicrobial fluoroquinolones induce, with strongly varying frequency, life-threatening hypoglycemias, which is explained by their ability to block K(ATP) channels in pancreatic B-cells and thus to initiate insulin secretion. In apparent contradiction to this, we observed that none of the fluoroquinolones in this study (gatifloxacin, moxifloxacin, ciprofloxacin, and a number of fluorophenyl-substituted compounds) initiated insulin secretion of perifused mouse islets when the glucose concentration was basal (5mM). Only when the glucose concentration was stimulatory by itself (10mM), the fluoroquinolones enhanced secretion. The fluoroquinolones were ineffective on SUR1 Ko islets, which do not have functional K(ATP) channels. All of these fluoroquinolones depolarized the membrane potential of mouse B-cells (patch-clamping in the whole-cell mode). Using metabolically intact B-cells (perforated-patch mode) however, 100microM of gatifloxacin, ciprofloxacin or moxifloxacin were unable to depolarize when the glucose concentration was 5mM, whereas other K(ATP) channel blockers (tolbutamide and efaroxan) remained effective. Only at a very high concentration (500microM) gatifloxacin and moxifloxacin, but not ciprofloxacin induced repetitive depolarizations which could be antagonized by diazoxide. In the presence of 10mM glucose all fluoroquinolones which enhanced secretion markedly elevated cytosolic calcium concentration ([Ca(2+)](i)). In the presence of 5mM glucose gatifloxacin and moxifloxacin at 500microM but not at 100microM elevated [Ca(2+)](i). It is concluded that fluoroquinolones in the clinically relevant concentration range are not initiators, but rather enhancers of glucose-induced insulin secretion. The block of K(ATP) channels appears necessary but not sufficient to explain the hypoglycemic effect of fluoroquinolones.