Here we discuss members of a new family of serine/threonine protein kinases with a likely role in cell cycle control. These kinases are referred to as polo-like kinases, after the prototypic founding member of the family, the polo gene product of Drosophila melanogaster. The polo kinase was originally identified in mutants that display abnormal mitotic spindle organization. Subsequently, potential homologues of Drosophila polo have been identified in yeasts (Cdc5p in Saccharomyces cerevisiae; plo1⁺ in Schizosaccharmoyces pombe) and in mammals (polo-like kinase 1; Plk1). Genetic and biochemical studies suggest that polo, Cdc5p and plo1⁺ may be required for mitotic spindle organization and, possibly, for cytokinesis. Likewise, the patterns of expression, activity and subcellular localization of Plk1 strongly suggest that this mammalian kinase functions also during mitosis, possibly in spindle assembly and function. In addition to Plk1, however, more distantly related members of the polo-like kinase family have been identified in mammalian cells, and the available data are consistent with the idea that some of these may act earlier in the cell cycle, possibly during G1. If this hypothesis is correct, different members of the pololike kinase family would act at several points during the cell cycle, reminiscent of the behaviour of Cdk/cyclin complexes.