We have measured by indirect ELISA the binding of neutralizing and non-neutralizing anti-lactate dehydrogenase-elevating virus (LDV) polyclonal and monoclonal antibodies to synthetic peptides representing unmodified hydrophilic segments of LDV proteins. Using this method a single neutralization epitope has been shown to be located in the very short (about 30 amino acid long) ectodomain of the primary envelope glycoprotein, VP-3P, encoded by ORF 5. Although the neutralization epitopes of neuropathogenic and non-neuropathogenic LDVs differ slightly in amino acid sequences, the neutralizing antibodies bind strongly to the epitopes of both groups of viruses. However, the neutralization epitopes of neuropathogenic and non-neuropathogenic LDVs are associated with different numbers of polylactosaminoglycan chains (1 and 3, respectively) which may affect the binding of neutralizing antibodies to the virions of these LDVs. The ELISA using synthetic peptides containing the neutralization epitope provides a novel, rapid, sensitive, and inexpensive method for quantitating LDV neutralizing antibodies in infected mice.