Infusion of hemoglobin-based blood substitutes results in an increase in blood pressure, primarily due to the binding of nitric oxide, the constituent of endothelium-derived relaxing factor (EDRF). Consequently, these blood substitutes could prove efficacious in the treatment of gram-negative septic shock, where overproduction of EDRF leads to severe, life-threatening hypotension. However, the effectiveness of these cell-free hemoglobin preparations in the treatment of septic shock may be limited by the interaction of bacterial lipopolysaccharide (LPS) with hemoglobin which leads to enhanced cellular toxicity. We examined how cell-free hemoglobin (HbAo) alters LPS-induced damage of endothelial cells in the presence and absence of serum in order to distinguish between LPS effects mediated by soluble CD 14 (a high affinity receptor for LPS present in serum) and LPS effects only observed at higher LPS concentrations. In the presence of 10% FBS (a source of soluble CD 14), LPS induced a dose-dependent inhibition of proliferation in bovine aortic endothelial cells (BAEC) with complete (100%) inhibition at 10 ng LPS/mL. LPS in the presence of serum also induced a dose-dependent increase in BAEC apoptosis which plateaued at 10 ng/mL, but the maximal extent of apoptotic cell death was only 45% ± 7% (mean ± SD, n=4). In both assays, the addition of HbAo had significant, protective effects on LPS induced apoptosis, inconsistent with a role for HbAo as an LPS-binding protein. In the absence of serum, LPS had no effect on BAEC apoptosis up to concentrations of 1 g/mL, but enhanced the apoptosis induced by HbAo. At 1 g/mL, LPS doubled the extent of apoptosis induced by 50 M HbAo (20% ± 6% wo. LPS vs 39% ± 9% w. LPS; n = 5). The enhanced apoptosis observed with LPS and HbAo combined was completely blocked by adding 0.4 mM Trolox®, an antioxidant. Trolox® had no effect on apoptosis induced by HbAo alone, or on the apoptosis induced by LPS in the presence of serum. In the presence of serum and at low LPS concentrations, conditions where LPS effects are predominantly mediated by soluble CD 14, HbAo had either no effect or protected against LPS induced cytotoxicity. Only in the absence of serum did HbAo combine with higher LPS concentrations to generate enhanced cytotoxicity. The protective effect of an antioxidant suggests that LPS may interact with hemoglobin to enhance the production of cytotoxic, oxidative species.