Bovine respiratory syncytial (BRS) virus can be divided into antigenic subgroups based on the reactivity of monoclonal antibodies (mAbs) to the attachment glycoprotein, G. Further, the polyclonal antibody response of calves vaccinated with recombinant vaccinia viruses expressing the G protein of a particular subgroup is also subgroup-specific. To investigate the genetic basis for the antigenic heterogeneity of the BRS virus G protein, the genes for the G protein from 6 BRS virus strains representative of the antigenic subgroups were cloned, sequenced, and compared with the prototype subgroup A strain, 391-2. There was only 10% nucleic acid difference and 15% amino acid difference between strains from different subgroups. These findings are in sharp contrast to the situation with human RS virus, where there is a 45% difference in amino acid identity between subgroups. In fact, the extent of amino acid difference between BRS virus subgroups is similar to the level of heterogeneity observed within human subgroups. Analysis of the reactivity of mAbs with peptides from the cysteine-rich region (174-188) of the G protein representing each antigenic subgroup indicated that amino acids at positions 180, 183, and possibly 184 are important in subgroup distinction. Taken together, these data suggest that although the genetic variation responsible for the antigenic differences determining subgroups among BRS viruses is more limited than that observed among human RS virus subgroups, the amino acid differences that exist have a profound effect upon antibody recognition.