A new, nonenzymatically treated preparation of amphibian sarcolemmal blebs has been used to study the regulation of skeletal muscle ATP-sensitive K+ [K(ATP)] channels. When a frog skeletal muscle fiber is split in half in a Ca(2+)-free relaxing solution, large hemispherical membrane blebs appear spontaneously within minutes without need for Ca(2+)-induced contraction or enzymatic treatment. These blebs readily formed gigaseals with patch pipettes, and excised inside-out patches were found to contain a variety of K+ channels. Most prominent were K(ATP) channels similar to those found in the surface membrane of other muscle and nonmuscle cells. These channels were highly selective for K+, had a conductance of approximately 53 pS in 140 mM K+, and were blocked by internal ATP. The presence of these channels in most patches implies that split-fiber blebs are made up, at least in large part, of sarcolemmal membrane. In this preparation, K(ATP) channels could be rapidly and reversibly blocked by glibenclamide (0.1-10 microM) in a dose-dependent manner. These channels were sensitive to ATP in the micromolar range in the absence of Mg. This sensitivity was noticeably reduced in the presence of millimolar Mg, most likely because of the ability of Mg2+ ions to bind ATP. Our data therefore suggest that free ATP is a much more potent inhibitor of these channels than MgATP. Channel sensitivity to ATP was significantly reduced by ADP in a manner consistent with a competition between ADP, a weak inhibitor, and ATP, a strong inhibitor, for the same inhibitory binding sites. These observations suggest that the mechanisms of nucleotide regulation of skeletal muscle and pancreatic K(ATP) channels are more analogous than previously thought.