Starvation, diabetes and insulin did not alter the concentration of casein kinases in rat liver cytosol. However, the K(m) for casein of casein kinase 2 from diabetic rats was about 2-fold lower than that from control animals. Administration of insulin to control rats did not alter this parameter, but increased the K(m) for casein of casein kinase 2 in diabetic rats. Starvation did not affect the kinetic constants of casein kinases. The effect of diabetes on casein kinase 2 persisted after partial purification of the enzyme by glycerol-density-gradient centrifugation and affected also its activity on other protein substrates such as phosvitin, high-mobility-group protein 14 and glycogen synthase. The results indicate that rat liver cytosol casein kinase 2 is under physiological control.