Sphingomyelin (SM) and cholesterol (Chol) are the major lipids in the mammalian cells, which are mainly localized to the plasma membrane. Multiple lines of evidence suggest that these lipids form local lipid domains in the plasma membrane, playing functional roles in the cell. Several observations have suggested that these lipid domains are required for cytokinesis. In this chapter, we show the methods for visualizing SM-rich and/or Chol-rich membrane domains at cytokinesis by using specific lipid-binding proteins. Lysenin, equinatoxin II, perfringolysin O, and pleurotolysin A2 bind specifically to clustered SM-rich domain, dispersed SM-rich domain, Chol-rich domain, and SM/Chol mixtures, respectively. Nontoxic forms of these lipid-binding proteins fused to fluorescent proteins are used for imaging lipid domains in biological membranes at cytokinesis. The image analysis reveals the structures and functions of SM-rich and/or Chol-rich domains at the time of cytokinesis.