Immunization with recombinant OspA protein of Borrelia burgdorferi protects against experimental Lyme disease. In the present study, mice were injected intramuscularly with plasmid DNA (VR2210) encoding strain B31 OspA. In this vector, the ospA-coding sequence was under transcriptional control of the cytomegalovirus immediate early promoter. For negative and positive controls, mice were immunized with either the plasmid vector without an osp-coding sequence or recombinant OspA protein, respectively. Mice immunized with VR2210 DNA produced OspA-specific antibodies that bound to B. burgdorferi in a whole cell ELISA and inhibited the growth of a homologous strain of B. burgdorferi. Immunization with VR2210 protected mice against challenge with 2 infectious strains of B. burgdorferi, Sh-2-82 and N40. These results indicate that vaccination with plasmid DNA expressing OspA is an efficacious method for providing a protective response against B. burgdorferi infection.