A set of antisera specific for each viral polypeptide of foot-and-mouth disease virus was used to provide a full comparison of polypeptides of two strains attenuated for cattle with respect to their parental virulent strains. Both attenuated strains, belonging to serotypes O1 Campos and C3 Resende, were obtained through serial passages of the corresponding virulent strains in chicken embryos. Although mutations were scattered throughout the genome, both attenuated strains showed an electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of viral polypeptide 3A faster than that of their respective wild-type strains. To determine the nature of this alteration, the nucleotide sequences of the genomic region encoding this polypeptide were determined. Comparative sequence analysis of wild-type and attenuated strains revealed 57 and 60 nucleotide deletions in the attenuated strains O1 Campos and C3 Resende, respectively. These studies, in conjunction with our previous analysis of recombinant viruses between wild-type and attenuated strains, which concluded that the major determinants of attenuation are located in the 3' half of the viral genome, strongly suggest that the alteration in polypeptide 3A of the attenuated strains is important for their reduced virulence in cattle.