alpha-Cells secrete glucagon in a fuel-dependent fashion. We tested the hypothesis that alpha-cells contain sulfonylurea- and ATP-sensitive K+ channels. We studied two clonal lines of alpha-TC cells (simian virus 40 T-antigen induced glucagonoma cells) and for reference purposes, similarly transformed beta-TC insulinoma cells. alpha-TC cells each contained approximately 3000 high-affinity binding sites for the sulfonylurea [3H]glyburide. Whole-cell ATP- and tolbutamide-sensitive K+ currents of alpha-TC and beta-TC cells, relative to cell surface area, were comparable. In cell-attached membrane patches of alpha-TC cells, two types of K+ channels were observed. They had slope conductances of approximately 63 and 33 pS when the electrode contained 151 mM K+. Tolbutamide and diazoxide decreased and enhanced, respectively, the open probability of these channels. The membrane of alpha-TC cells depolarized periodically. This electrical activity was inhibited by diazoxide. A physiological mixture of amino acids enhanced glucagon release, and high glucose partially inhibited this release. Tolbutamide also enhanced glucagon release, whereas diazoxide inhibited it. Thus, alpha-TC glucagonoma cells contain ATP-sensitive K+ channels that regulate glucagon release, yet allow inhibition of hormone release by glucose.