We have previously demonstrated that the nonspecific binding of unfractionated heparin (UFH) to plasma proteins has a marked modulating effect on its anticoagulant activity. Since some heparin-binding proteins are also acute-phase-reactant proteins, we explored the possibility that the induction of the acute-phase response can increase the plasma concentrations of heparin-binding proteins. The recovery of a fixed amount of UFH or low-molecular-weight heparin (LMWH) added in vitro to rat plasma samples obtained at various time intervals after the administration of intravenous endotoxin or subcutaneous turpentine was compared with that of saline-treated control animals. The anti–factor Xa activity was measured in the plasma samples before and after the addition of a chemically modified low-affinity heparin (LAH) to displace the proportion of the added heparin that is reversibly bound to plasma proteins. Our results show that at 6 hours post–endotoxin and at 24 hours post–turpentine treatment, virtually no anti–factor Xa activity could be measured in the plasma samples, while the expected levels were obtained for control plasma. After the addition of LAH to displace protein-bound UFH, essentially the same anti–factor Xa levels were measured in the plasma from all three treatment groups. These results indicate that induction of the acute-phase reaction can dramatically increase the levels of heparin-binding proteins in rat plasma. In addition, we compared the anti–factor Xa recovery of UFH with that of an LMWH from the plasma of endotoxin- and saline-treated rats and demonstrated that LMWH binds less to plasma proteins than UFH, even in plasma in which the levels of heparin-binding proteins are markedly elevated. The recovery of a fixed amount of UFH added in vitro to human plasma from septic patients was also reduced, but not to the same extent as seen in rat plasma. Removal of candidate heparin-binding and acute-phase proteins by immunodepletion indicated that vitronectin plays an important role in the nonspecific binding of UFH in patient plasma.