The first serum specimens obtained after onset of illness and those obtained later from patients with infectious mononucleosis were examined for neutralization of P3HR-1-derived Epstein-Barr virus (EBV). Without complement, viral neutralization indices of sera obtained soon after onset were all less than 0.2, and those of sera obtained later were less than 0.5. In contrast, sera from EBV-seropositive, healthy adults had neutralization indices of 0.45-1.0. Sera from EBV-seronegative adults showed no evidence of viral neutralization. The addition of complement from EBV antibody-negative human serum to 31 early and 34 late serum samples from 34 patients with infectious mononucleosis yielded neutralization indices of greater than or equal to 0.95. Complement-dependent neutralizing antibodies were also demonstrated in sera from EBV-seropositive, healthy adults. In contrast, sera from EBV-seronegative adults failed to neutralize EBV after the addition of complement. Heat inactivation destroyed the capacity of complement to induce additional neutralization of EBV by sera from patients with mononucleosis. The early appearance of complement-dependent neutralizing antibodies in the course of mononucleosis is consistent with a role in recovery from acute EBV infection.