A panel of monoclonal antibodies (MAbs) to BHV-1, specific for gI and gIII glycoproteins and for a nonglycosylated core protein p100, was used to examine the epitope specificity of the immune response to the virus in naturally exposed and experimentally infected cattle. Sera from experimentally infected calves were analyzed in a competition ELISA (C-ELISA). Antibody to an epitope on gIII appeared as early as 4 days post infection, although virus-neutralizing antibody did not appear until day 8 or later. Antibody production peaked at 13 to 18 days post infection but the level of antibody to each gIII epitope varied. Competition by sera from cattle naturally exposed to BHV-1 was analyzed as a function of the virus neutralization (VN) titer of these sera. Competition with most of the MAbs correlated linearly with neutralization titer. However, competition with 2 of the MAbs, one specific for gIII and one specific for gI, was maximal even at the lowest neutralization titer, and competition with another MAb, specific for a non-glycosylated core protein, p100, was negative. Selected sera from the naturally exposed group were also examined by radioimmunprecipitation, and were shown to react predominantly with gI, gIII and gIV glycoproteins and in a few shown to react predominantly with gI, gIII and gIV glycoproteins and in a few MAbs were determined, and it was found that neutralization was enhanced by certain combinations. A mutually exclusive relationship between neutralization enhancement and competition for binding by MAbs (as determined by reciprocal C-ELISA) indicated an epitope-specific, rather than antibody-specific, mechanism for neutralization enhancement.