A panel of 26 monoclonal antibodies (mAbs) against glycoprotein B (gB) of Aujeszky's disease (pseudorabies) virus (ADV), a glycoprotein complex consisting of three glycoproteins, gBa, gBb, and gBc, was produced by two research groups and was used for the topographical epitope mapping of gB. An epitope map was constructed in which the identified epitopes of gB were situated in 14 topologically distinct antigenic domains; ten antigenic domains represented by 22 mAbs were localized on gBc, while four antigenic domains represented by four mAbs resided on gBb of the gB complex. All the epitopes located on gBc appeared to be conformation-dependent, whereas all the epitopes on gBb were conformation-independent. The identified epitopes of gB were conserved among laboratory, vaccine and field ADV strains. Conformation-dependent epitopes were shown to contribute largely to the overall antibody response to gB in naturally infected swine and immunized mice. Moreover, it was found that most of the infected animals responded relatively weakly to the identified conformation-independent epitopes of gB, while a group of immunodominant epitopes that induced a strong antibody response was represented exclusively by conformation-dependent epitopes from different antigenic domains. The results clearly demonstrated that conformation-dependent epitopes of gBc play a crucial role in inducing the humoral immune response to gB of ADV during the natural infection of swine and immunization of mice. The application of mAbs of our panel as research and diagnostic tools is discussed.