Recently we demonstrated that normal polyspecific immunoglobulin given intravenously (IVIG) and plasma samples from patients treated with IVIG neutralize the mitogenic and cytokine-inducing activities of group A streptococcal (GAS) superantigens. Here we investigated whether this neutralizing activity is mediated by antibodies to these superantigens. IVIG and plasma samples collected from a patient with GAS necrotizing fasciitis post-IVIG infusions markedly inhibited the mitogenic activity elicited by the streptococcal pyrogenic exotoxins SpeB and SpeC, as well as by GAS culture supernatant. Immunoblot analysis showed marked increases in the levels of antibodies to SpeC and proteins in the GAS culture supernatant in post-IVIG over those of pre-IVIG plasma samples. Removal of antisuperantigen antibodies in IVIG by adsorption to SpeC- and GAS culture supernatant-coupled Sepharose markedly reduced the neutralizing ability of IVIG against respective stimuli. The neutralizing activity was totally recovered in the eluted antibodies. By contrast, although pre- and post-IVIG plasma samples contained antibodies to SpeA, these antibodies did not block the activity of this superantigen. Nonspecific immunomodulatory activity of IVIG was ruled out because neither the IVIG nor the affinity-purified antibodies significantly inhibited the response to the polyclonal T-cell mitogen phytohemagglutinin A. These data provide direct evidence that the neutralizing activity in IVIG, and in patient plasma samples following IVIG treatment is mediated by antibodies to superantigens and indicate that the quality rather than the quantity of these antibodies may be more clinically relevant.