Crystalline hemoglobin solution was lyophilized following deoxygenation or addition of several compounds, or both, to establish protective conditions for obtaining freeze-dried hemoglobin chemically and functionally unaltered and clinically suitable as a blood substitute. Glucose and sucrose were most active in protecting the hemoglobin molecule from deterioration. The results of stability studies demonstrated that lyophilized hemoglobin maintained at 4 degrees C. did not show any significant alteration in structure and function for a period of nine months. Freeze-dried hemoglobin samples stored at room temperature were unchanged for six months, but after this time, a progressive increase in methemoglobin content and a decrease in P50 were observed. The effectiveness of lyophilized hemoglobin in vivo was investigated by transfusions in rats exchanged to blood replacements of 75 or 95 per cent, using lyophilized hemoglobin reconstituted soon after lyophilization or after seven months of storage at room temperature. The data show that lyophilized, reconstituted hemoglobin is effective in restoring or maintaining, or both, vital signs. In rats transfused to a 75 per cent blood replacement, several hematologic and physiologic parameters change soon after transfusion but return to normal pretransfusion levels within seven days after transfusion.