Observations in the early 1990s that plasmid DNA could directly transfect animal cells in vivo sparked exploration of the use of DNA plasmids to induce immune responses by direct injection into animals of DNA encoding antigenic proteins. This method, termed DNA immunization, now has been used to elicit protective antibody and cell-mediated immune responses in a wide variety of preclinical animal models for viral, bacterial, and parasitic diseases. DNA vaccination is particularly useful for the induction of cytotoxic T cells. This review summarizes current knowledge on the vectors, immune responses, immunological mechanisms, safety considerations, and potential for further application of this novel method of immunization.