The identification of antigens with the capacity to induce a broad spectrum of protective immunity is an important consideration in the design of a Lyme disease vaccine. In this study, the range of protection provided by outer surface protein (Osp) A or OspC vaccination was compared. Mice actively immunized with OspA or OspC were challenged with 3 North American isolates of Borrelia burgdorferi. OspA-immunized mice were fully protected from infection with each of the isolates, whereas mice immunized with OspC were protected from infection with the homologous isolate but not with 2 heterologous isolates. Sequence analysis revealed that the ospA genes from these 3 isolates were >99% homologous, whereas the ospC genes shared only 81%-85% homology. Western blot analysis suggested antigenic heterogeneity associated with OspC but not OspA. These results indicate that genetic and antigenic heterogeneity may limit the usefulness of OspC as a vaccine constituent.