Neutralizing antibody responses to human immunodeficiency virus type 1 (HIV-1) vary widely and have not been reproducibly associated with prognosis or disease progression. We have found that both low-passage clinical isolates and laboratory-adapted strains of HIV-1 have different sensitivities to neutralization by the same antiserum, depending on the host cell in which the viral stock is prepared. One such isolate (VL069) grown in H9 cells was neutralized by 20 human sera at a geometric mean titer of 1:2,047; this same isolate prepared in peripheral blood mononuclear cell (PBMC) culture was neutralized at a mean titer of < 1:10 by the same sera. Adsorption and mixing experiments indicated that neither antibody to H9 cell components nor blocking by excess viral antigen was responsible for the differences observed. This host cell effect is rapidly reversible upon passage of the virus from PBMCs to H9 cells and back into PBMCs. In contrast, the neutralization characteristics remained remarkably stable over extended culture in PBMCs. Two laboratory strains and five clinical isolates were evaluated in expanded studies of this phenomenon. While the neutralization characteristics of most of the strains studied were affected by the host cell in which the strain was propagated, two of the strains (one clinical isolate and one laboratory strain) appeared antigenically unaffected by their cell of origin. Host cell effect was also evident in neutralization by monoclonal antibodies directed against the CD4-binding region and the V2, V3, and gp41 regions. Possible mechanisms for this host cell effect include (i) mutation during passaging; (ii) selection in different host cells of different subpopulations of the (uncloned) viral stock; and (iii) cell-specific posttranslational modifications. To explore these possibilities, the V3 through V5 region of gp120 was sequenced in preparations made by passing VL069 into H9 cells and into PBMCs; HIVMN grown in CEM-SS cells and in PBMCs was also sequenced. In both cases, a few amino acid changes outside the V3 region were found. Studies are currently under way to assess the significance of these changes.