Succinylated human serum albumin (Suc-HSA) was synthesized by treating human serum albumin with succinic anhydride. Among similar proteins and neo(glyco)proteins tested, Suc-HSA exhibits a pronounced net negative charge, a feature that largely contributes to its efficacy against replication of human immunodeficiency virus type 1 (HIV-1). To assess further the antiviral effect of Suc-HSA, the effect on HIV-1 replication was studied in the presence of whole human plasma. Pretreatment of MT2 cells with Suc-HSA was more efficacious than direct Suc-HSA treatment of HIV prior to addition to the cells. No changes in the antiviral effect of Suc-HSA were observed in tissue culture medium, 30% plasma, or whole plasma when CPDA-1 (citrate-phosphate-dextrose-adenine 1) was used as the anticoagulant. However, a dramatic decrease (greater than 99%) in the antiviral activity was observed when these experiments were performed in plasma prepared from blood using heparin as anticoagulant. The antagonistic effect by heparin was observed both in the case that heparin was added prior to or after addition of Suc-HSA to the test system. In the present study we demonstrate that heparin largely reduces Suc-HSA activity on HIV replication in the same concentration in which if affects binding of Suc-HSA to the envelope protein gp120 and in particular its V3 domain. In the same concentration range, heparin reduced binding of Suc-HSA to MT4 cells, another HTLV-I-transformed cell line. It is concluded that heparin can displace Suc-HSA from its binding sites on hybrid lymphoid cells as well as on HIV-1 particles. Therefore, we conclude that both the binding to cells and to virus contribute to the potent anti-HIV-1 effect. The fact that heparin and heparin degradation products antagonize Suc-HSA without having a significant anti-HIV-1 effect indicates that the anticoagulant acts as a relatively weak partial inhibitor.