Earlier studies suggest that glitazones exert beneficial effects in patients with type 2 diabetes by directly affecting insulin secretion of β-cells, besides improving the effectiveness of insulin in peripheral tissues. The effects of glitazones on stimulus-secretion coupling (SSC) are poorly understood. We tested the influence of troglitazone and pioglitazone on different parameters of SSC, including insulin secretion (radioimmunoassay), cell membrane potential, various ion currents (patch-clamp), mitochondrial membrane potential (ΔΨ), and cytosolic Ca(2+) concentration (fluorescence). Troglitazone exerted stimulatory, inhibitory, or no effects on insulin secretion depending on the drug and glucose concentration. It depolarized the ΔΨ, thus lowering ATP production, which resulted in opening of ATP-dependent K(+) channels (K(ATP) channels) and reduced insulin secretion. However, it also exerted direct inhibitory effects on K(ATP) channels that can explain enhanced insulin secretion. Troglitazone also inhibited the currents through voltage-dependent Ca(2+) and K(+) channels. Pioglitazone was less effective than troglitazone on all parameters tested. The effects of both glitazones were markedly reduced in the presence of bovine serum albumin. Glitazones exert multiple actions on β-cell SSC that have to be considered as undesired side effects because the influence of these compounds on β-cells is not controllable. The final effect on insulin secretion depends on many parameters, including the actual glucose and drug concentration, protein binding of the drug, and the drug by itself. Troglitazone and pioglitazone differ in their influence on SSC. It can be assumed that the effects of pioglitazone on β-cells are negligible under in vivo conditions.