A cyclic GMP-dependent protein kinase was previously found in the 0.3 M NaCl extract of avian liver nucleoli [1]. The kinase phosphorylates preferentially a protein of a molecular weight of approximately 11,000 present in calf thymus histone mixture (type IIA, Sigma) and in isolated liver nucleoli. Further studies with purified protein substrates have now indicated that the chromatin-associated protein, which is preferentially phosphorylated by the cyclic GMP-dependent kinase, is high mobility group protein HMG 14. Histone H1 was also a relatively good phosphate acceptor but in this case the phosphorylation was not cyclic GMP-dependent and therefore due to a different protein kinase present in the partially purified nucleolar extract. Acid hydrolysis of the phosphorylated HMG 14 and subsequent analysis by chromatography and high-voltage electrophoresis indicated that the phosphorylated amino acid residue in HMG 14 is phosphoserine.