An in situ gel kinase assay was used to resolve kinase activities during the HeLa cell cycle. Three kinase activities changed dramatically during the cell cycle, whereas a number of others remained relatively constant. The former included histone kinases with estimated molecular masses of 46, 87, and 39 kDa. The 46-kDa kinase activity was preferentially expressed at least 10-fold greater in mitotic cells. In contrast, the 87- and 39-kDa kinases were most active early in G1 but their activity declined progressively as cells approached mitosis. The 87- but not the 46- or 39-kDa kinases had autophosphorylation activity. The identities of the kinases are unknown but they may play important roles in regulation of the cell cycle.