Polynucleotide immunization has been described as the Third Revolution in Vaccinology. Early studies suggest the potential benefits of this form of immunization including: long-lived immunity, a broad-spectrum of immune responses (both cell mediated immunity, and humoral responses) and the simultaneous induction of immunity to a variety of pathogens through the use of multivalent vaccines. Using a murine model, we studied methods to enhance and direct the immune response to polynucleotide vaccines. We demonstrated the ability to modulate the magnitude and direction of the immune response by co-administration of plasmid encoded cytokines and antigen. Also, we clearly demonstrated that the cellular components (cytosolic, membrane-anchored, or extracellular) to which the expressed antigen is delivered determines the types of immune responses induced. Since induction of immunity at mucosal surfaces (route of entry for many pathogens) is critical to prevent infection, various methods of delivering polynucleotide vaccines to mucosal surfaces have been attempted and are described. Expansion of studies in various species, using natural models, should be extremely helpful in demonstrating the universality of this approach to immunization and more importantly, accurately identify parameters that are critical for the development of protective immunity.