The potential targets of marine sulfated polymannuroguluronate (SPMG) involved in inhibition of HIV-1 entry were investigated by surface plasmon resonance and flowcytometry. Results indicated that binding of SPMG either to soluble oligomeric rgp120 or to complexed rgp120–sCD4 mainly resided in V3 loop region. In addition,SPMGwas shown to be less accessible for sCD4 when sCD4 had pre-interacted with rgp120, though SPMG per se multivalently bound to sCD4 with relatively low affinity. While the pre-incubation of SPMG with rgp120 caused a partial blockade of rgp120 binding to sCD4, suggesting that SPMG either shared common binding sites on gp120 with sCD4 or masked the docking sites of gp120 for sCD4. Taken together, V3 domain was demonstrated to be the major site mediating interaction of SPMG with complexed rgp120–sCD4. It seems likely that SPMG binds to both rgp120 and sCD4, but has less accessibility for sCD4 when sCD4 has already bound to rgp120. Nevertheless, addition of SPMG either prior to or after the interaction of rgp120 with sCD4 may suppress rgp120 binding to sCD4. The exact pattern of this trimolecular complex formation at the cell membrane-anchored virus level requires further clarification.