We tested for the presence of sulfonylurea receptors in pancreatic alpha cells. Two high affinity sulfonylurea receptors were identified in clonal pancreatic alpha cells (alpha TC-6): a 140-kDa species observed previously in clonal pancreatic beta cells (HIT) and a second 150-kDa protein. The dissociation constant (Kd) for both receptors is approximately 3.5 nM for an iodinated glyburide analog, 5-iodo-2-hydroxyglyburide. The estimated number of receptors (Bmax) increases approximately 2-fold, from 3.1 to 6.8 pmol/mg of membrane protein as the pH of the binding buffer is reduced from 7.5 to 6. Consistent with the notion that high affinity sulfonylurea receptors are integral components of the ATP-sensitive K+ channel, we demonstrated the presence of ATP-sensitive K+ channels in inside-out patches of alpha TC-6 cells. Whole cell K+ currents that activated with time showed inward rectification at positive potentials (above 0 mV) and were almost completely suppressed by 5 nM glyburide. Likewise, glyburide blocked 86Rb+ efflux from ATP-depleted alpha TC-6 cells, an effect that was reversed by 400 microM diazoxide. The presence of sulfonylurea receptors provides a mechanism by which sulfonylureas can directly modulate alpha cell function. The properties of the 150-kDa receptor and the role of ATP-sensitive K+ channels in alpha cells remain to be elucidated, but as in beta cells, ATP-sensitive K+ channels may be involved in metabolic regulation of alpha cells by glucose.