Mitotic HeLa cells showed an increased phosphorylation activity towards myelin basic protein compared to cells in G1 or S phases. Further investigation using renaturation gels revealed that, in mitotic cell lysates, a protein with an apparent molecular mass of around 40 kDa phosphorylates myelin basic protein. This kinase is active early in mitosis, but is then downregulated concomitantly with p34cdc2 kinase as mitosis proceeds, its activity decreasing to basal levels by early G1. The molecular mass of the kinase suggested that it might be one of the human homologues of rat erk1 or erk2. However, antibodies raised against C-terminal sequences of erk1 and erk2 failed to immunoprecipitate renaturable kinase activity from mitotic lysates. In addition, in immunoblots erk1 and erk2 failed to show the well established changes in electrophoretic migration that are consequences of their activation. These data indicate that these two mitogen-activated protein (MAP) kinases are not stimulated during HeLa cell mitosis and indicate that the 40-kDa kinase is either a new member of the MAP kinase family or it is a novel mitotic kinase that has not yet been described.