The peptide Arg-Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu was shown to be a specific substrate for casein kinase II (CK II) in extracts of 3T3-L1 cells. Fractionation of a cell extract on DEAE-cellulose revealed only one peptide kinase and it eluted at the same salt concentration required to elute CK II. Consistent with the properties of CK II, the peptide kinase activity was inhibited by very low concentrations of heparin (Ki less than 6 nM) and it used GTP efficiently as a substrate. A Western blot, developed with antiserum to bovine thymus CK II, demonstrated the presence of CK II protein in 3T3-L1 extracts and that peptide kinase activity was directly related to the amount of CK II protein. The peptide was used to assay CK II activity in extracts of 3T3-L1 cells stimulated to differentiate into adipocytes. Differentiation produced a transient increase in CK II activity that reached a maximum (4-fold) on day 4. The increased activity was accounted for by increased CK II protein. Induction of CK II preceded the increase in total protein and was not the result of cell proliferation. CK II induction was coincident with induction of the insulin receptor, but, whereas insulin binding remained elevated, CK II activity declined after day 4. Agents that stimulate differentiation of 3T3-L1 cells did not cause induction of CK II in 3T3-C2 cells that do not differentiate. The transient nature of the induction of CK II suggests that the kinase may contribute to the process of differentiation rather than being a phenotypic change like that of the insulin receptor.