Polyanionic compounds are known to inhibit the binding of human immunodeficiency virus (HIV) to CD4+ cells and the subsequent fusion step between the virus and cells. We selected an HIV-1 strain resistant to dextran sulfate (DS) by cultivation of HIV-1 (NL4-3)-infected MT-4 cells in the presence of DS Mr 5000. DS did not inhibit the binding of DS-resistant virus to MT-4 cells or syncytium formation between MOLT cells and HUT-78 cells persistently infected with the DS-resistant virus. In addition, a monoclonal antibody with specificity for the V3 loop of envelope gp120 glycoprotein did not recognize the DS-resistant HIV-1 gp120 V3 loop. The following mutations were found in the gp120 molecule of the DS-resistant HIV-1 strain but not in the wild-type strain: S114N in the V1 loop region; S134N in the V2 loop region; K269E, Q278H, and N293D in the V3 loop region; N323S in the C3 region; a deletion of five amino acids (Phe-Asn-Ser-Thr-Trp) at positions 364-368 in the V4 loop; and R3871 in the CD4 binding domain. Our results suggest that (i) DS interacts with specific amino acid residues in the gp120 molecule, (ii) the virus is able to overcome the inhibitory effect of DS on viral infectivity, (iii) cross-resistance developed against those polyanionic compounds that are structurally related to DS, and (iv) the molecular determinants of HIV cell tropism, syncytium-inducing ability, coreceptor (fusin/ CC-CKR5) utilization, and polyanion resistance seem to be located in the env genome of HIV and specifically in the V3 loop domain.