The V3 loop of the human immunodeficiency virus (HIV) surface protein, gp 120, constitutes a principal neutralizing determinant. HIV strains lacking a naturally conserved N-linked oligosaccharide (at position 306) within the V3 loop are highly sensitive to neutralization. We subjected molecular clones of HIV(LAI) lacking this 306N-glycan to in vitro immune selection with MAbs directed against the V3 loop. In all, ten clones were characterized, and all proved resistant to V3-directed neutralization. Sequencing of the V3 loop revealed that six of the clones had become resistant at least partly by reacquisition of the 306N-glycan. Only two of the clones possessed mutations within the binding site of the antibody itself, while the two remaining clones did not display changes within the V3 loop itself. Thus, HIV strains lacking the 306N-glycan primarily develop resistance to V3-directed neutralization through acquisition of the specific oligosaccharide. This demonstrates that protein glycosylation can be a primary modifier of virus antigenicity of possible importance for the interaction of HIV with the host immune response.