Hemoglobin-based oxygen carrier-201 (HBOC-201, hemoglobin glutamer-250 [bovine], Hemopure®; Biopure Corporation, Cambridge, MA) is polymerized hemoglobin of bovine origin being developed as an oxygen therapeutic. In this study, we evaluated the tolerability of a single intraoperative dose of HBOC-201 in surgical patients. In a single-blinded, multicenter study, 81 patients were randomized to receive either a single infusion of HBOC-201 (55 patients) or an equivalent volume of lactated Ringer's solution (26 patients). Forty-two patients originally assigned to the HBOC-201 group received the entire planned treatment of only one of the following doses: 0.6, 0.9, 1.2, 1.5, 2.0, or 2.5 g/kg of body weight. Thirteen of the 55 patients in the HBOC-201-assigned group did not reach the trigger point for transfusion administration, and they were not included in the analysis. We studied clinical outcomes and compared hematologic findings, blood chemistry values, and blood use in the two treatment groups. There were no patient deaths in this study. No pattern of clinically significant laboratory abnormalities could be attributed to exposure to HBOC-201. In the HBOC-201 group, 2 patients had a transient increased concentration of serum transaminases and 6 had transient skin discoloration. One patient in the HBOC-201 group had mast cell degranulation with hypotension. Postoperatively, methemoglobin plasma concentrations increased in the HBOC-201 group in a dose-dependent manner, reaching maximal values of 3.7% ± 3.2% (average of all doses given) on postoperative day 3. There was no difference in the mean number of allogeneic blood units transfused in the 2 groups (3.3 ± 1.8 and 3.7 ± 4.1 for the lactated Ringer's solution and HBOC-201 groups, respectively) over the course of hospitalization. The intraoperative administration of HBOC-201, up to a maximum of 245 g, was generally well tolerated. There was no relationship between HBOC-201 use and the number of allogeneic blood units transfused over the entire hospitalization course. The administration of HBOC-201 was associated with a delayed (third postoperative day) dose-dependent increase in the plasma methemoglobin concentration. We conclude that the intraoperative use of HBOC-201 was generally well tolerated.