The objective of the present study was to demonstrate the usefulness of recombinant adenoviral vector in the generation of monoclonal antibodies (MAb) against natural epitopes of proteins using the glycoprotein gC of pseudorabies virus (PRV) as the target antigen. The recombinant adenovirus expressing the glycoprotein gC (Ad-gC) was constructed according to the AdMax method. Three immunization protocols consisting of various combinations of intramuscular injection of Ad-gC and a plasmid DNA expressing gC (pcDNA-gC) were conducted in BALB/c mice at 2-week intervals. The two groups with the highest antibody levels (Ad-gC/Ad-gC and pcDNA-gC/pcDNA-gC/Ad-gC) were selected for fusion following a final protein boost. Nine MAbs against the glycoprotein gC of PRV were subsequently developed and characterized to be isotypes of IgG1, IgG2a, and IgG2b with ascitic titers ranging from 1:2 x 10(5) to 1:5 x 10(6). Immunofluorescence assay (IFA) and Western blotting analysis confirmed that these MAbs could recognize linear epitopes on the glycoprotein gC of PRV. Our results provide a new strategy for preparation of specific MAb against viral protein.