A protein kinase activity has been identified in regenerating rat liver, which can not be separated from topoisomerase II activity throughout all purification steps including glycerol gradient sedimentation. The protein was estimated about 125KDa in size as determined by glycerol gradient sedimentation analysis. SDS-PAGE revealed two bands of 55KDa and 67KDa. A 40KDa band was also identified in another purification method. Despite the different sizes, they showed the same functional aspect except autophosphorylation. The protein kinase activity absolutely required magnesium ion, and this requirement was not substituted by other mono and divalent ions. The activity showed a high affinity for histone H1 as a substrate. Both activities of topoisomerase II and protein kinase were completely inactivated by novobiocin, a topoisomerase II inhibitor, at a concentration of 1-5mM. No similar activity has previously been identified in regenerating rat liver.