The synthetic hexapeptide Ser-Glu-Glu-Glu-Val-Glu and its N-acetylated derivative are readily and specifically phosphorylated by rat liver casein kinase TS (type-2), while the derived heptapeptide with an additional N-terminal Arg is a very poor substrate. Conversely, the substitution of Glu for Val5 in the synthetic peptide Arg-Arg-Ser-Thr-Val-Ala, which is a good substrate for cAMP-dependent protein kinase by virtue of the N-terminal arginyl residues, prevents its phosphorylation by this enzyme. These data indicate that the site specificities of these two classes of protein kinases, requiring acidic and basic residues on the C- and N-terminal sides of the target residue(s), respectively, are mutually incompatible.