Immunoglobulin G fractions from patients with Lambert-Eaton myasthenic syndrome (LEMS), an autoimmune disease of neuromuscular transmission, immunoprecipitate 125I-labeled omega-conotoxin GVIA-labeled calcium channels solubilized from rat brain. A 58-kDa antigen was detected by probing Western blots of partially purified calcium channels with LEMS plasma and IgG and was shown to be the relevant antigen in omega-conotoxin receptor immunoprecipitation. Monoclonal antibody 1D12, produced by immunizing mice with synaptic membranes, has properties similar to these autoimmune IgGs in both immunoprecipitation and Western blotting assays. 1D12 antigen was purified by immunoaffinity chromatography and shown to bind LEMS IgG. The antigen was identified by screening a rat brain cDNA library with 1D12 and was found to have strong homology to the synaptic vesicle membrane protein synaptotagmin. Our results indicate therefore that these antibodies immunoprecipitate omega-conotoxin receptors by binding to synaptotagmin that is associated with calcium channels. We suggest that the interaction between synaptotagmin and the voltage-gated calcium channel plays a role in docking synaptic vesicles at the plasma membrane prior to rapid neurotransmitter release and that autoantibody binding to a synaptotagmin-calcium-channel complex may be involved in the etiology of LEMS.