Dialysis of human red blood cells using a hypotonic solution and a commercial kidney dialysis unit followed by ultrafiltration through 0.1 micron pore hollow fibers provides an easily managed method for isolation of lipid-free hemoglobin. High pressure liquid chromatography analysis of lipid-free hemoglobin (LFHB) indicates 99-100% protein purity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrated that LFHB migrates as a single band. The process requires hypoosmotic dialysis of human RBC to a final 119-139 (av 132) mosmol/kg osmotic pressure. Additional reduction in osmotic pressure results in irreversible cell lysis which results in lipid contamination of the hemoglobin. Processing one-half liter of packed red blood cells requires 10 h, resulting in an average of 90% hemoglobin recovery.