Insulin treatment significantly altered the elution profile of deproteinized muscle extracts chromato-graphed on Sephadex G-25 columns, particularly in fraction II, which contains the insulin mediator. Further purification of fraction II by high-voltage paper electrophoresis at pH 1.9 and 3.5 resulted in two active fractions. Fraction 1 → 4 stimulated the cyclic AMP-dependent protein kinase and inhibited glycogen synthase phosphoprotein phosphatase, and may be a novel substance. Fractions 1 → > 6 and 3 → 6 inhibited the cyclic AMP-dependent protein kinase and stimulated glycogen synthase phosphatase. It is proposed that the insulin mediator is present in fractions 1 → 6 and 3 → 6.