1. Islets from normal mice were used to study the mechanisms by which imidazoline antagonists of alpha 2-adrenoceptors increase insulin release in vitro. 2. Alinidine, antazoline, phentolamine and tolazoline inhibited 86Rb efflux from islets perifused with a medium containing 3 mM glucose, i.e. under conditions where many adenosine 5'-triphosphate (ATP)-sensitive K+ channels are open in the beta-cell membrane. They also reduced the acceleration of 86Rb efflux caused by diazoxide, an opener of ATP-sensitive K+ channels. 3. ATP-sensitive and voltage-sensitive K+ currents were measured in single beta-cells by the whole-cell mode of the patch-clamp technique. Antazoline more markedly inhibited the ATP-sensitive than the voltage-sensitive current, an effect previously observed with phentolamine. Alinidine and tolazoline partially decreased the ATP-sensitive K+ current. 4. The four imidazolines reversed the inhibition of insulin release caused by diazoxide (through opening of ATP-sensitive K+ channels) or by clonidine (through activation of alpha 2-adrenoceptors) in a concentration-dependent manner. Only the former effect correlated with the ability of each drug to increase control insulin release stimulated by 15 mM glucose alone. 5. It is concluded that the ability of imidazoline antagonists of alpha 2-adrenoceptors to increase insulin release in vitro can be ascribed to their blockade of ATP-sensitive K+ channels in beta-cells rather than to their interaction with the adrenoceptor.