The effect of T3 was studied on phosphorylation of cytosolic proteins in rat anterior pituitary. Cytosols obtained from hypothyroid (H) and T3 (50 micrograms/100 g body weight) injected H rats (T) were phosphorylated in vitro. Although total incorporation of 32P into proteins did not differ between H and T, analysis of phosphoproteins by SDS polyacrylamide gel electrophoresis and autoradiography revealed that T3 injection significantly increased phosphorylation of 2 proteins within 1 h. These results and our previous demonstration of T3 effects on phosphorylation in rat liver cytosol and cultured human skin fibroblasts suggest that protein phosphorylation is an important early intracellular process in T3 action.