The role of carbohydrate in the antigenic and immunogenic structure of bovine herpesvirus type 1 (BHV-1) glycoproteins gI and gIV was investigated. Deglycosylated proteins induced a significantly lower antibody response in rabbits than native glycoproteins suggesting that the immunogenicity of several epitopes on gI and gIV is carbohydrate-dependent. Loss of carbohydrate from gI also resulted in a significantly decreased ability to induce a serum neutralizing antibody response to BHV-1, due to modifications in three distinct carbohydrate-containing continuous epitopes. Similarly, in vitro lysis of BHV-1-infected cells was significantly reduced when antibodies raised against deglycosylated gI were employed; this was attributed to changes in two of the three carbohydrate-dependent neutralizing epitopes on gI. The oligosaccharides may be directly involved as actual components of these continuous epitopes, rather than in stabilization of the conformation of the protein. In contrast, carbohydrate removal from gIV did not have a significant effect on the capacity to stimulate a neutralizing antibody response. Accordingly, none of the neutralizing epitopes on gIV appeared to be carbohydrate-dependent. Similarly, lysis of virus-infected cells was not significantly reduced when antibodies specific for deglycosylated rather than native gIV were used. In contrast to the humoral response, the delayed-type hypersensitivity response was stronger in rabbits immunized with deglycosylated proteins than in those inoculated with native glycoproteins gI or gIV. Consequently, the carbohydrates on gI and gIV may play a dual role in the host's immune recognition and response by contributing to certain epitopes, but masking others. The implications for the development of a subunit vaccine against BHV-1 are discussed.