We used cultured myotubes to demonstrate complement-dependent lysis of muscle membranes by serum from patients with myasthenia gravis. Lysis was monitored by light microscopy and release of incorporated [86Rb]. In the presence of guinea pig complement (GPC), 18 of 37 heat-inactivated myasthenic sera (49%), but none of 16 controls, caused morphologically detectable myotube lysis. Ten of 19 myasthenic sera (53%) increased [86Rb]-release compared with 10 controls. Immunoglobulin fractions retained the complement-dependent lytic activity. Inactivation of GPC prevented the lysis. [86Rb]-release appeared to correlate with clinical severity. The complement-dependent lysis resulted in a decrease in the number of acetylcholine receptors (AChRs) in myotubes, and AChR-immunoglobulin complexes were found in the medium of lysed cultures. The data suggest that cultured myotubes can be used to document complement-dependent antibody reactions in the pathogenesis of myasthenia.