Five fractions of phosphoprotein kinase activity were separated by phosphocellulose chromatography of nuclear non-histone phosphoproteins isolated from exponentially-growing HeLa S3 cells. Each fraction contained a heterogeneous group of proteins and possibly more than one protein kinase. These fractions were characterized with respect to specific activity and optimal concentrations of Mg2+ and Mn2+. The ability of each kinase fraction to catalyze the incorporation of 32P from [γ-32P]ATP into various molecular weight classes of heat-inactivated non-histone nuclear phosphoproteins (as substrate) was examined by electrophoretically resolving the proteins in SDS-polyacrylamide gels. Each kinase fraction induced a distinctive pattern of protein phosphorylation. Substrate preferences (non-histone phosphoprotein or histone) were determined for each fraction by examining the incorporation of 32P from [γ-32P]ATP into both TCA-precipitable material and into specific molecular weight classes of proteins. The activities of two of the fractions to phosphorylate non-histone nuclear phosphoprotein were stimulated by the presence of histones. Studies were undertaken to assess the effects of cAMP or cGMP on the activity of each kinase fraction. No significant sensitivity to the cyclic nucleotides was observed for any of the kinase fractions.