Sera from patients with Lyme disease were evaluated for their ability to kill Borrelia burgdorferi in vivo and in vitro. Separate groups of C3H mice received sera from seropositive humans with early- or late-stage Lyme disease or from seronegative controls. Eighteen to 24 hours after passive transfer of sera, the mice were challenged with 100,000 low-passage B. burgdorferi strain B31 or CA287 organisms. Sera from subjects with late-stage Lyme disease protected the mice against infection after challenge with B. burgdorferi, but sera from subjects with early-stage Lyme disease were not protective. Late-stage sera also inhibited the growth of B. burgdorferi in microcultures on Barbour-Stoenner-Kelly media better than early-stage sera. Immunoblot analysis revealed that the protective properties of late-stage sera were associated with a response of antibodies to multiple proteins. This response included strong reactivity with the outer-surface proteins A and B, which was lacking in early-stage sera.