Chromosomal high mobility group (HMG) proteins have been examined as substrates for cGMP-dependent and cAMP-dependent protein kinases. Of the four HMG proteins only HMG 14 contained a major high affinity site which could be phosphorylated by both enzymes, preferentially by cGMP-dependent protein kinase. One mol of 32P was incorporated/mol of HMG 14. Kinetic analysis revealed apparent Km and Vmax of 40.5 microM and 14.7 mumol/min/mg, respectively, for cGMP-dependent protein kinase, and 123 microM and 11.1 mumol/min/mg, respectively, for cAMP-dependent protein kinase. Tryptic maps of 32P-labeled phosphopeptides of HMG 14 demonstrated phosphorylation of the same site by both enzymes. The tryptic fragment containing the major phosphorylation site was identified by amino acid composition and sequence as HMG 14 (residues 4-13): H-Lys-Val-Ser(P)-Ser-Ala-Glu-Gly-Ala-Ala-Lys-OH. HMG 14 and HMG 17 also contained minor sites which could be phosphorylated by cGMP-dependent protein kinase. Tryptic phosphopeptides mapping suggested that the same minor site was phosphorylated on both HMG 14 and 17. On the basis of amino acid composition, the tryptic peptides carrying the minor phosphorylation sites were identified as H-Leu-Ser(P)-Ala-Lys representing residues 23-26 and 27-30 of HMG 14 and HMG 17, respectively.