It is generally recognized that DNA vaccines are often less effective in large animals than in mice. One possible reason for this reduced effectiveness may be transfection deficiency and the low level of expression elicited by plasmid vectors in large animals. In our attempt to enhance transfection efficiency and, thereby, enhance immune responses, we employed a variety of methods inducing gene gun delivery or suppositories as delivery vehicles to mucosal surfaces, as well as electroporation for systemic immunization. To test these different systems, we used two different antigens-a membrane antigen from bovine herpesvirus glycoprotein (BHV-1) gD and a particulate antigen from hepatitis virus B. Gene gun and suppository delivery of BHV-1 gD to the vagina resulted in the induction of mucosal immunity not only in the vagina, but also at other mucosal surfaces. These data support the contention of a common mucosal immune system. In the case of electroporation, we were able to develop significant enhancement of gene expression following electroporation with surface electrodes (non-invasive electroporation) as well as invasive electroporation using single or six-needle electrodes. Various delivery systems such as bioject or needle delivery also influenced the immune response in both the presence and absence of electroporation. These studies also demonstrated that co-administration of plasmids coding for two different antigens (BHV-1 gD and hepatitis B surface antigen (HbsAg)) did not result in significant interference between the plasmids. These studies suggest that various combinations of delivery systems can enhance immunity to DNA-based vaccines and make them practical for administration of these vaccines in large animals.