Over 100 preclinical studies in several small and large animal species were performed to evaluate the safety and efficacy of diaspirin crosslinked hemoglobin (DCLHb; Baxter Healthcare Corp.) as an oxygen therapeutic. During the preclinical evaluation of DCLHb, myocardial lesions were observed following the administration of DCLHb to certain species. These lesions were characterized as minimal to moderate, focal-to-multifocal myocardial degeneration and/or necrosis that were scored using a severity scale of minimal to marked in relative severity. The lesions were typically observed 24–48 h after single topload infusions of DCLHb into rhesus monkeys or pigs at doses as low as 200 or 700 mg/kg, respectively. Dogs, sheep, and rats did not develop these lesions after single-dose administrations of DCLHb. The left ventricular myocardium, typically near the base of or including the papillary muscles, was the most severely affected region, followed by the intraventricular septum and the right ventricle. The left and right atria were usually not affected. In a study in rhesus monkeys, morphometric analysis revealed that these lesions comprised less than 3% of the total myocardium. Although increases in serum enzyme activities (AST, CK, LDH) were observed after infusion of DCLHb, myocardial-related isoenzymes did not increase. ECG analysis and echocardiography were not altered by these lesions, and there was no observable adverse effect on myocardial function. Polymerization of DCLHb reduced, but did not eliminate, the incidence and severity of the lesions. However, infusion of hemoglobin solutions with reduced reaction rates with nitric oxide (NO) resulted in a significant decrease in lesion incidence and severity, while administration of L-NAME, an NO synthase inhibitor, resulted in the appearance of lesions that were indistinguishable from those induced by hemoglobin, suggesting that reduction in normal NO levels is an important mechanistic factor. Overall, the presence of myocardial lesions represents a histopathologic finding that must be considered during the preclinical testing and development of new HBOCs.