1. The patch clamp technique has been used to characterize the effects of phentolamine, an unselective blocker of alpha 1- and alpha 2-adrenoceptors, on the electrical activity of isolated RINm5F insulin-secreting cells and the gating of ATP-regulated potassium (K+ATP) channels. 2. Current-clamp experiments carried out by use of both conventional whole-cell recordings and nystatin-perforated cells, have demonstrated that phentolamine (5-20 microM) in the complete absence of alpha-adrenoceptor agonists, caused a sharp depolarization of the cell membrane from approximately -66 mV to -42 mV. This depolarization was associated with the generation of calcium action potential-like spikes. In the continued presence of phentolamine, diazoxide (100 microM) reversed these effects by causing a hyperpolarization of the cell, thereby preventing Ca2+ spikes. 3. Unitary current events from K+ATP channels were recorded from both outside-out membrane patches and saponin permeabilized or open-cells. When added to either the inside or the outside of the plasma membrane, phentolamine (0.1-100 microM) blocked openings from these channels. The effects of phentolamine were rapid, sustained and fully reversible. Phentolamine was apparently a more effective blocker of channels from the inside than the outside of the membrane. 4. The KI value, corresponding to 50% inhibition of channels was estimated to be approximately 0.7 microM when phentolamine was added to the inside of the membrane and the Hill coefficient approximately 1. 5. Yohimbine (1-10 microM) and the chemically 2-substituted imidazoline alpha-adrenoceptor antagonists, antazoline (25 microM) and tolazoline (25 microM) were also found to block K+ATP channels in isolated patches of membrane. 6. In conclusion the present study demonstrates that phentolamine and other imidazoline adrenoceptor antagonists have effects upon ATP-sensitive K+ channels that are not associated with stimulation of the adrenoceptor.