1. The effects of phentolamine and yohimbine on adenosine 5'-triphosphate (ATP)-sensitive K+ channels were studied in normal mouse beta-cells. 2. In the presence of 3 mM glucose, many ATP-sensitive K+ channels are open in the beta-cell membrane. Under these conditions, phentolamine inhibited 86Rb efflux from the islets. This inhibition was faster with 100 than with 20 microM phentolamine but its steady-state magnitude was similar with both concentrations. Yohimbine (20-100 microM) also inhibited the efflux rate but was not as potent as phentolamine. 3. In the presence of 6 mM glucose, most ATP-sensitive K+ channels are closed in the beta-cell membrane. Their opening by 100 microM diazoxide caused a marked acceleration of 86Rb efflux from the islets. This acceleration was almost entirely prevented by 20 microM phentolamine. It was barely affected by 20 microM yohimbine and reduced by 50% by 100 microM yohimbine. 4. ATP-sensitive K+ currents were studied in single beta-cells by the whole cell patch-clamp technique. Phentolamine (20-100 microM) caused a progressive but almost complete and irreversible inhibition of the current. The effects of yohimbine were faster but smaller; the inhibition was still incomplete with 100 microM yohimbine. 5. The increase in ATP-sensitive K+ current produced by 100 microM diazoxide was prevented by 100 microM phentolamine but only partially attenuated by 100 microM yohimbine. 6. It is concluded that phentolamine inhibits ATP-sensitive K+ channels in pancreatic beta-cells. This novel effect of phentolamine resembles that of hypoglycaemic sulphonylureas. It may account for previously unexplained effects of the drug. These observations also call for reinterpretation of many studies in which phentolamine was used as an allegedly specific blocker of alpha-adrenoceptors.