An assay for the neutralization of human immunodeficiency virus type 1 (HIV-1) is described in which the reduction in infectious titer of HIV-1 after preincubation at 37 degrees C with antibody-positive serum is the measure of neutralization. The assay format and its controls allow several experimental manipulations that, taken together, indicate an effect of antibody on HIV-1 infectivity that occurs before or independently of HIV-1 attachment. The direct inactivation of HIV-1 infectivity by antibody is irreversible and temperature dependent, requires a bivalent antibody directed against accessible envelope determinants, and does not require a heat-labile or (Ca2+)- or (Mg2+)-dependent cofactor. The mechanism of inactivation cannot be explained by agglutination of virus, nor is it associated with disruption or dissociation of envelope protein from virions. Rather, the antibody is likely to perturb some metastable property of the envelope that is required for entry. Laboratory-adapted HIV-1 isolates were more sensitive to the inactivating effects of sera than were primary patient isolates. The latter were particularly resistant to inactivation by contemporary autologous sera, a feature not explained by blocking antibodies. Additional studies showed a weak relationship between disease course and serum inactivation of the reference LAI laboratory strain of HIV-1. Heteroduplex analysis and autologous inactivation assays of sequential specimens from individual patients indicate that over time, the viral quasispecies that emerge and dominate are resistant to the inactivating effects of earlier sera.