Recently we identified a novel autoantigenic region in the alpha chain of the human acetylcholine receptor (HuAChR), residues 160-167 (P. L. Schwimmbeck, T. Dyrberg, D. B. Drachman, and M. B. A. Oldstone, J. Clin. Invest., in press). Antibodies to that sequence appeared in the sera of a subset of patients with myasthenia gravis (MG) and affinity-purified antibody was biologically active for an in vitro assay of the HuAChR. Sequence homology between the autoantigen and two separate regions of herpes simplex virus glycoprotein D (HSV-GpD) have been identified. These components are, respectively, the HuAChR, residues [160-167] (PESDQPDL), and residues [286-293] (PNATQPEL) and [381-388] (PEDDQPSS) of HSV-GpD. Antisera from rabbits immunized with synthetic peptides representing HSV-GpD [286-293] bound strongly to the AChR peptide. In contrast, antiserum to HSV-GpD [381-388] bound minimally, if at all, even though both virus peptides shared four amino acids with the receptor sequence. To investigate the molecular basis for the differential binding, we tested the reactivity of the HSV-GpD antisera to analogs of the [381-388] virus peptide containing single amino acid substitutions. The results demonstrated that the cross-reactivity between HuAChR [160-167] and HSV-GpD [286-293] predominantly depends on a single residue, the C-terminal leucine in HSV-GpD, position 293.