Several different preparations of cross-linked hemoglobin (CLHb) are being evaluated for their efficacy and safety as red cell substitutes in a variety of preclinical and clinical settings. Because CLHb is known to sequester nitric oxide (NO) and inhibit NO-mediated processes, we hypothesized that CLHb would have a hemostatic effect by enhancing platelet reactivity, inducing vasoconstriction, or both. Infusion of o-raffinose CLHb shortened the prolonged microvascular bleeding time and decreased blood loss from ear incisions in rabbits rendered anemic and thrombocytopenic. Moreover, this hemostatic effect persisted for at least 24 hours after infusion. Phenylephrine induced a degree of vasoconstriction similar to that induced by CLHb but did not shorten the bleeding time or decrease blood loss, suggesting that vasoconstriction alone cannot account for the hemostatic effect of CLHb. There was no evidence of CLHb-induced activation of coagulation in vivo, since infusion of CLHb did not increase circulating levels of thrombin-antithrombin complex. In vitro, CLHb abolished the inhibitory effect of the NO donor 3-morpholinosydnonimine on platelet aggregation and enhanced the aggregation of stimulated but not resting platelets. This potentiating effect was not attenuated by the addition of superoxide dismutase or catalase. To evaluate the potential arterial thrombogenicity of CLHb, a model of carotid artery thrombosis was developed in rabbits without thrombocytopenia or anemia. Compared with albumin infusion, CLHb infusion shortened the time to complete carotid occlusion. These data suggest that CLHb may shift the thromboregulatory balance toward clot formation, resulting in decreased bleeding in anemic and thrombocytopenic rabbits and possibly increasing arterial thrombogenicity in normal rabbits.