As an allosteric protein, hemoglobin can assume at least two different quatenary structures, a deoxy or T for tense conformation and an oxy or R for relaxed conformation (2,3) depending upon its state of ligation. Because the pK values of oxy and deoxy- hemoglobins are different, molecules in the R conformation should behave differently than molecules in the T conformation on ion exchange chromatography. Kilmartin et al. (4) designed an anaerobic cation exchange chromatographic procedure with which they separated the deoxy hemoglobin from oxy hemoglobin and other R conformation hemoglobin components like CO-hemoglobin and sulfohemoglobin. In this report we describe the further development of Kilmartin's procedure to separate two electro-phoretically silent hemoglobin mutants, Hb Potomac [β101 Glu→Asp] (5) a high oxygen affinity variant, and Hb M-Milwaukee [β67 Val→Glu] (6,8) a low oxygen affinity mutant from Hb A. The modifications we have made include (a) applying the hemolysate at a partial pressure of oxygen determined from an examination of the oxygen equilibrium curve to produce a favorable ratio of T to R conformations of the two hemoglobns to be separated, (b) controlling the partial pressure of oxygen in the chromatographic developer, and (c) monitoring the oxygen tension of the column effluent during the development of the chromatogram.Hb Potomac. This work has been supported by grants HL20142 and AMl785O from the National Institutes of Health, U.S. Public Health Service.