The pattern and sites of phosphorylation of high mobility group (HMG) chromosomal proteins in HeLa cells labeled in vivo with [32P]orthophosphate have been compared with those of isolated HeLa HMG protein labeled in vitro by purified protein kinase enzymes. In synchronized HeLa cells there is phosphorylation of two HMG proteins designated hHMG 14 alpha 1 and alpha 2. hHMG 14 alpha 1 and alpha 2 are phosphorylated in a single identical tryptic phosphopeptide which runs toward the anode with electrophoresis at pH 4.7. The specific activity of phosphorylation at this site increased 2.5-fold in both hHMG 14 alpha 1 and alpha 2 in metaphase compared to interphase cultures. In vitro only casein kinase II specifically catalyzed phosphorylation of hHMG 14 alpha 1 and alpha 2 among a mixture of hHMG proteins; phosphorylation occurred at the site which was phosphorylated in vivo. The site of phosphorylation catalyzed by casein kinase II is distinct from sites in HMG proteins phosphorylated by cyclic nucleotide-dependent protein kinases or by casein kinase I. Casein kinase I specifically catalyzed phosphorylation of histone H1. These results indicate that casein kinase II is the enzyme which catalyzes the major phosphorylation of hHMG protein which occurs in vivo.