Five distinct seroprotective groups among North American and European isolates of Borrelia burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii have been identified using the in vitro borreliacidal assay. The predominant North American seroprotective group comprised isolate 297 and B. burgdorferi isolates from California, Illinois, Wisconsin, New York, and Texas. A second group was represented by isolate C-1-11. The majority of European isolates belonged to a seroprotective group composed of B. garinii. Another European group contained isolates classified genetically as genospecies group VS461 (B. afzelii). A fifth group, represented by isolate LV5, could kill both North American and European isolates of B. burgdorferi sensu lato. These results suggest that combinations of immunogenic protective proteins of spirochetes will be necessary to provide a comprehensive vaccine.