During cytokinesis in eukaryotic cells, an actomyosin-based contractile ring (CR) is assembled along the equator of the cell. Myosin II ATPase activity is stimulated by the phosphorylation of the myosin II regulatory light chain (MRLC) in vitro, and phosphorylated MRLC localizes at the CR in various types of cells. Previous studies have determined that phosphorylated MRLC plays an important role in CR furrowing. However, the role of phosphorylated MRLC in CR assembly remains unknown. Here, we have used confocal microscopy to observe dividing HeLa cells expressing fluorescent protein-tagged MRLC mutants and actin during CR assembly near the cortex. Di-phosphomimic MRLC accumulated at the cell equator earlier than non-phosphorylatable MRLC and actin. Interestingly, perturbation of myosin II activity by non-phosphorylatable MRLC expression or treatment with blebbistatin, a myosin II inhibitor, did not alter the time of actin accumulation at the cell equator. Furthermore, inhibition of actin polymerization by treatment with latrunculin A had no effect on MRLC accumulation at the cell equator. Taken together, these data suggest that phosphorylated MRLC temporally controls its own accumulation, but not that of actin, in cultured mammalian cells.