Lyme disease is a chronic, multisystemic infection caused by Borrelia burgdorferi sensu lato. An infectious strain of B. burgdorferi was previously shown to bind to human platelets via the integrin alpha IIb beta 3. In this study, a diverse group of Lyme disease spirochetes was tested for platelet- and alpha IIb beta 3-binding activity. This collection included representatives of each of the three species that cause Lyme disease, B. burgdorferi (sensu stricto), B. garinii, and B. afzelii. Strains were characterized for infectivity in mouse models or were low-passage isolates from human patients. Each of the 11 infectious strains bound to platelets immobilized in microtiter wells and in suspension. Binding to platelets in suspension was specifically inhibited by a blocking anti-alpha IIb beta 3 antibody, and representatives of each species bound to purified alpha IIb beta 3. The strains that did not bind alpha IIb beta 3 or platelets were all noninfectious. No obvious relationship was observed between binding to platelets and expression of the bacterial outer surface protein OspA, OspB, or OspC, as assessed by immunoblotting. These results demonstrate that integrin alpha IIb beta 3-binding activity is widespread among the Borrelia species that cause Lyme disease and are consistent with a role for alpha IIb beta 3 binding in the transmission and/or pathogenesis of Lyme disease.