Sulfated polymannuroguluronate (SPMG), a marine sulfated polysaccharide, has entered the Phase II clinical trial in China as the first anti-acquired immune deficiency syndrome (AIDS) drug candidate obtained from marine organisms. To determine the binding site(s) (receptors) of SPMG in lymphocytes mediating its anti-AIDS activities, fluorescein-5-isothiocyanate (FITC)-labeled SPMG was used to investigate SPMG binding to lymphocytes. Flow cytometry (FCM) and fluorescence microscopy analysis showed that the SPMG binds to lymphocytes in a rapid, specific, reversible, and saturable fashion. Several SPMG binding proteins were purified by affinity chromatography from lymphocyte membrane preparations. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting analysis revealed that a 55 kDa lymphocyte membrane protein is CD4. To characterize the SPMG and CD4 interaction, inhibition assay and surface plasmon resonance (SPR) assay were carried out. SPMG bound to CD4 in a multivalent fashion with specificity. The binding of SPMG to human lymphocyte CD4 was competitively inhibited by human soluble CD4 (hsCD4). Likewise, the binding between hsCD4 and immobilized SPMG was blocked by excess free SPMG. These results indicate that CD4 is one of the specific SPMG binding sites (receptors) in lymphocytes. The interaction between SPMG and CD4 may provide a mechanistic explanation of the immunopotentiating and anti-AIDS activities of SPMG in human immunodeficiency virus (HIV) infected individuals.