Nateglinide and mitiglinide (glinides) are characterized as rapid-onset and short-acting insulinotropic agents. Although both compounds do not have a sulfonylurea structure, it has been postulated that insulin secretion is preceded by their binding to Kir6.2/SUR1 complex, and a mechanism of insulin secretion of glinides has been accounted for by this pathway. However, we hypothesized the involvement of additional mechanisms of insulin secretion enhanced by glinides, and we analyzed the pattern of time course of insulin secretion from MIN6 cells with the existence of agents that have specific pharmacologic actions. Dose-dependent effects of tolbutamide, glibenclamide, nateglinide, and mitiglinide were observed. Insulin secretion induced by 3 microM tolbutamide and 1 nM glibenclamide was completely inhibited by 10 microM diazoxide and 3 microM verapamil, although the latter half-component of insulin secretion profile induced by 3 microM nateglinide or 30 nM mitiglinide remained with the existence of those agents. Glinides enhanced insulin secretion even in Ca2+-depleted medium, and its pattern of secretion was same as the pattern with existence of verapamil. The latter half was suppressed by 1 microM dantrolene, and concomitant addition of verapamil and dantrolene completely suppressed the entire pattern of insulin secretion enhanced by nateglinide. Thus, we conclude that glinide action is demonstrated through two pathways, dependently and independently, from the pathway through K(ATP) channels. We also demonstrated that the latter pathway involves the intracellular calcium release from endoplasmic reticulum via ryanodine receptor activation.