Stroma-free hemoglobin (SFHb) is prepared from outdated blood. Red cells are washed, lysed in hypotonic phosphate buffer, and stromal lipid is then removed by toluene extraction and high-speed centrifugation. Pyridoxal-phosphate (PP) added in a 4:1 molar ratio to deoxygenated SFHb, is covalently linked across the polyphosphate binding site of the Hb tetramer by reduction with NaBH4 under N2 for 18 h. Excess reagents are removed by dialysis. Subsequent crosslinking using 5.0% glutaraldehyde in the presence of lysine for 12-36 h yields soluble, macromolecular polyhemoglobin (PolyHb). Progress of the reaction is monitored by gel chromatography. In vitro work shows that different molecular weight (MW) PolyHb can be produced, ranging in weight from 130,000 to over 1,000,000. Intermediate MW PolyHb (less than 600,000) remains stable for months at 4 degrees C, withstands prolonged incubation at 37 degrees C, and has a viscosity similar to blood. SFHb has a low P50 = 15 torr, which has been raised to 26 torr by pyridoxylation. When PP-SFHb is crosslinked into PP-PolyHb, it can reversibly carry oxygen with a lower affinity (P50 = 16 torr) than nonpyridoxylated PolyHb (P50 = 11 torr). These favorable physicochemical properties of PP-PolyHb suggest that further progress toward a potential artificial blood substitute has been made.