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Background: The assembly of an F-actin- and myosin-II-containing contractile ring (CR) is required for cytokinesis in eukaryotic cells. Interactions between myosin II and actin in the ring are believed to generate the force that constricts the cell into two daughters. The mechanism(s) that contribute to the spatially and temporally regulated assembly and disassembly of the CR at the cell equator are poorly understood., Results: We generated an LLCPK1 epithelial cell line that stably expresses GFP-actin. Live confocal imaging showed accumulation of GFP-actin in the equatorial cortex from late anaphase through cytokinesis. Fluorescence recovery after photobleaching (FRAP) experiments showed that actin in the CR is highly dynamic (t(1/2) = 26 s). In some cells, movement of GFP-actin toward the equatorial region was observed and contributed to FRAP. Blocking actin dynamic turnover with jasplakinolide demonstrates that dynamic actin is required for CR formation and cytokinesis. To test the role of myosin II in actin turnover and transport during CR formation, we inhibited myosin light-chain kinase with ML7 and myosin II ATPase activity with blebbistatin. Inhibition of myosin light-chain phosphorylation resulted in clearance of GFP-actin from the equatorial region, a reduction in myosin II in the furrow, and inhibition of cytokinesis. Treatment with blebbistatin did not block CR formation but reduced FRAP of GFP-actin and prevented completion of cytokinesis., Conclusions: These results demonstrate that the majority of actin in the CR is highly dynamic and establish novel roles for myosin II in the retention and dynamic turnover of actin in the CR.