DNA-based vaccines represent a novel method of immunization that has been demonstrated to induce immune responses in animals against a variety of plasmid encoded antigens and following a number of different methods of vaccine delivery. We characterized the immune response to DNA-based vaccines encoding intracellular, membrane anchored (cell associated) and extracellular (secreted) forms of glycoprotein D (gD), an antigen from the viral envelope of the bovine herpesvirus-1 (BHV-1). Intramuscular injection of mice with plasmids encoding secreted or cell associated forms of this antigen led to seroconversion and a predominance of splenic IFN ã. Mice receiving plasmids encoding cell associated or secreted antigens displayed a predominance of IgG2a and IgG 1, respectively. The predominant serum isotype correlated with the cytokine and antibody isotype profiles within the draining lymph node. We demonstrated modulation of immune responses in mice following co-delivery of plasmids encoding a secreted form of gD and each of eight different murine cytokines (IL-1á, IL-12, IL-4, IL-6, IL-10, GM-CSF, IFN ã, TNF á). Plasmids encoding GM-CSF, TNF á, IL-4 and IL-6 demonstrated the capacity to enhance serum IgG titers and seroconversion efficiency. Plasmids encoding IFN ã and TNF á increased levels of serum IgG2a in mice. Varying the dose of plasmids encoding GM-CSF enhanced (10 [mu]g) or suppressed (50 [mu]g) serum antibody levels and induced significant increases in IL-4 levels in the spleen and draining lymph nodes. High doses of GM-CSF (50 [mu]g) increased the levels of serum IgG2a after boosting. Co-administration of plasmids encoding IFN ã either reduced (10 [mu]g) or enhanced (50 [mu]g) serum antibody levels and elevated mean serum IgG 2a levels. Finally, we investigated the potential for plasmids encoding the secreted form of gD to elicit immune responses in passively immune mice. We demonstrated that a single intramuscular immunization of passively immune C3H.HeN or C57BL/6 mice with plasmids encoding the secreted form of BHV-1 gD resulted in the development of both cell-mediated and humoral immunity.