It is now widely accepted that cancer arises at least partly due to the perturbation of normal cell cycle progression, in which reversible protein phosphorylation plays an important regulatory role. In addition to the well-documented cyclin-dependent kinases, the Polo-like kinase (Plk) family has emerged as a key player in many cell cycle related events. Genetic and biochemical experiments with several different organisms have demonstrated that polo kinases are involved in many aspects of mitosis, such as mitotic entry, sister chromatids separation and cytokinesis. To investigate the function of Plk1 in mammalian cells in more detail, we recently reported the phenotype of cultured cells after Plk1 depletion using RNA interference (RNAi). Plk1 depletion in cancer cells induces G2/M arrest, followed by apoptosis. A close correlation between mammalian Plk1 expression and carcinogenesis was recently documented. Overexpression of Plk1 was observed in various human tumors, such as melanomas, non-small cell lung cancer, etc. Constitutive expression of Plk1 in normal cells causes oncogenic focus formation and induces tumor growth in nude mice. Thus, inhibition of Plk1 function may be an important application for cancer therapy. Because inhibition of cell proliferation and reaction of apoptosis are basic principles in anticancer therapy, it will be intriguing to test the effect of Plk1 depletion in normal primary cells. A lentivirus-based RNAi approach is currently being used to deplete Plk1 in several types of normal cells. Using a yeast two-hybrid system, we have identified a battery of potential Plk1-interacting proteins, and the significance of these interactions during cell cycle progression will be analyzed.