Growth-associated H1 histone kinase, a homolog of the yeast cdc2+/CDC28 protein kinases that control entry into mitosis, is a chromatin-bound cyclic nucleotide-independent enzyme found only in growing cells. In a procedure involving salt extraction of chromatin, ammonium sulfate precipitation, and three chromatographic steps, the enzyme has been purified greater than 10,000-fold from Novikoff hepatoma cells. Enzyme purified by this procedure catalyzes the transfer to H1 histone of 2.7 mumol of phosphate/min/mg, a specific activity within the range of those reported for a number of homogeneous or nearly homogeneous protein kinases. Further purification to near homogeneity was achieved by an additional step of sucrose density gradient fractionation. Enzyme activity in the sucrose gradient is associated with two polypeptides of apparent Mr 60,000 and 33,000 on sodium dodecyl sulfate-gel electrophoresis. Substrate specificity studies show that in addition to H1, proteins with H1-like structure and function including histone H1(0), the erythrocyte-specific H5 histone, and the testis-specific H1t histone are phosphorylated. Nucleosome core histone H3, high mobility group proteins 1, 2, 14, and 17, protamine, casein, and ribosomal protein S6 are not substrates.