Notexin is a neurotoxic and myotoxic phospholipase A2 derived from the venom of the Australian tiger snake, Notechis scutatus. Though the phospholipase activity has been implicated in the toxicity of notexin, little is understood of its site and mode of action. In this study we investigated the myotoxicity of notexin on the skeletal muscle of the rat. Using immunogold labeling both in vivo and in vitro, we demonstrated that notexin bound exclusively to the sarcolemma. At the early stages when notexin was injected into the muscle there was no evidence of internalization, though at more advanced degeneration when muscle fibers were necrotic, the toxin was able to penetrate the interior of the fibers to exhibit nonspecific labeling. We also showed the toxin was able to bind to glycolytic muscle fibers, which are known to be resistant to the myotoxic effects of notexin. Electron microscopy confirmed the localization of the binding site to the sarcolemma. Scanning electron microscopy showed that the primary pathological changes associated with exposure to notexin were the appearance of areas of hypercontraction along the muscle fibers associated with small lesions in the sarcolemma. At more advanced stages large tears appeared in the sarcolemma between adjacent areas of hypercontraction, revealing apparently intact myofibrils below. We conclude that the sarcolemma is the binding site for the toxin. We propose that the hydrolytic activity causes the appearance of small lesions in the membrane, the loss of ion gradients, and hypercontraction. The weakened area between sites of hypercontraction is then ruptured, leading ultimately to the degeneration of the muscle fibers.