A nonhistone chromosomal protein (D-55) of Mr 55000 has been isolated in homogeneous form from calf thymus by using the standard salt-extraction procedures for the isolation of nonhistone chromosomal proteins followed by hydroxyapatite chromatography. D-55 is further characterized as coming from the group of nonhistone chromosomal proteins easily phosphorylated by an endogenous nuclear protein kinase. The kinase incorporates 1 mol of phosphate per mol of protein from [gamma-32P]ATP. The unphosphorylated form of D-55 binds to DNA, histones, and nucleosomes. Phosphorylation of D-55 does not significantly alter the binding of D-55 to DNA but greatly enhances its binding to histones and nucleosomes. Binding of D-55 to reconstituted nucleosomes enhances transcription of the nucleosome DNA by E. coli RNA polymerase by approximately 100-fold, to a level approximately 4 times that observed with naked calf thymus DNA as template. Phosphorylation of D-55 abolishes this enhancement. Binding of D-55 produces no apparent alteration in nucleosome structure as assayed by nuclease digestion patterns. In contrast, phospho-D55 alters nucleosome structure significantly.