Cyclin-dependent protein kinases are among the key regulators of eukaryotic cell cycle progression. Potential functions of the five cdc2-related kinases (CRK) in Trypanosoma brucei were analyzed using the RNA interference (RNA(i)) technique. In both the procyclic and bloodstream forms of T. brucei, CRK1 is apparently involved in controlling the G(1)/S transition, whereas CRK3 plays an important role in catalyzing cells across the G(2)/M junction. A knockdown of CRK1 caused accumulation of cells in the G(1) phase without apparent phenotypic change, whereas depletion of CRK3 enriched cells of both forms in the G(2)/M phase. However, two distinctive phenotypes were observed between the CRK3-deficient procyclic and bloodstream forms. The procyclic form has a majority of the cells containing a single enlarged nucleus plus one kinetoplast. There is also an enhanced population of anucleated cells, each containing a single kinetoplast known as the zoids (0N1K). The CRK3-depleted bloodstream form has an increased number of one nucleus-two kinetoplast cells (1N2K) and a small population containing aggregated multiple nuclei and multiple kinetoplasts. Apparently, these two forms have different mechanisms in cell cycle regulation. Although the procyclic form can be driven into cytokinesis and cell division by kinetoplast segregation without a completed mitosis, the bloodstream form cannot enter cytokinesis under the same condition. Instead, it keeps going through another G(1) phase and enters a new S phase resulting in an aggregate of multiple nuclei and multiple kinetoplasts in an undivided cell. The different leakiness in cell cycle regulation between two stage-specific forms of an organism provides an interesting and useful model for further understanding the evolution of cell cycle control among the eukaryotes.