To elucidate whether phosphorylation of myosin II regulatory light chain (MRLC) is essential for myosin II recruitment to the furrow during cytokinesis, HeLa cells transfected with three types of GFP-tagged recombinant MRLCs, wild-type MRLC, non-phosphorylated form of MRLC, and phosphorylated form of MRLC, were examined. Living cell-imaging showed that both phosphorylated and non-phosphorylated form of MRLCs were recruited to the equator at the same time after anaphase onset, suggesting that phosphorylation of MRLC is not responsible for recruitment of myosin II to the equator. Moreover, the treatment with an inhibitor of myosin II activity, blebbistatin, induced no effect on recruitment of those three recombinant MRLCs. During cytokinesis, phosphorylated but not non-phosphorylated form of MRLC was retained in the equator. These results suggest that phosphorylation of MRLC is essential for retainment of myosin II in the furrow but not for initial recruitment of myosin II to the furrow in dividing HeLa cells.