Phosphoenolpyruvate carboxykinase has been purified by a procedure involving chromatography on norleucine-Sepharose, using a decreasing (NH4)2 SO4-inorganic phosphate concentration gradient, followed by chromatography on GMP-Sepharose. The enzyme is eluted from the affinity column with 2.5 X 10(-5)m IDP. The same procedure is applicable to hog mitochondrial and cytosol enzymes yielding the enzyme as a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The corresponding molecular weight is 65,000 for each enzyme. Discontinuous polyacrylamide gel electrophoresis of the mitochondrial enzyme gave two sharp bands that were found to contain carboxykinase activity upon assay of gel slices. Similar analysis of the cytosol enzyme showed a much different staining pattern and four regions of carboxykinase activity. Plots of relative mobility of the two mitochondrial bands vs. per cent polyacrylamide in the gels gave parallel lines, confirming that the two bands did not result from enzyme aggregation. Elution of the enzyme upon analytical Sephacryl S-200 chromatography did, however, produce some resolution of the two bands without change in specific activity. The purification procedure described above has been applied also to the human mitochondrial enzyme with similar results. The molecular weight, similarly is 68,000.