This paper describes the derivation and characterization of monoclonal antibodies reactive with the group-specific epitopes of the VP1 peptide shared by enteroviruses. When tested against a wide range of the prototype strains of Coxsackie viruses A and B, polioviruses, and echoviruses, the monoclonal antibodies reacted with all of them in two or more assay systems. The virus of hepatitis A was the only enterovirus tested which was not recognized by these monoclonal antibodies. This finding is consistent with recent reports demonstrating genomic dissimilarity between this virus and the other enteroviruses. Although monoclonal antibodies have frequently been found to be assay specific, these particular antibodies were found to be equally effective in different immunochemical and biological assay systems. Biochemical characterization of one of these monoclonal antibodies is described and its potential usefulness for diagnosis is discussed.