Cholesterol plays important roles in biological membranes. The cellular location where cholesterol molecules work is prerequisite information for understanding their dynamic action. Bioimaging probes for cholesterol molecules would be the most powerful means for unraveling the complex nature of lipid membranes. However, only a limited number of chemical or protein probes have been developed so far for cytological analysis. Here we show that fluorescently-labeled derivatives of theonellamides act as new sterol probes in mammalian cultured cells. The fluorescent probes recognized cholesterol molecules and bound to liposomes in a cholesterol-concentration dependent manner. The probes showed patchy distribution in the plasma membrane, while they stained specific organelle in the cytoplasm. These data suggest that fTNMs will be valuable sterol probes for studies on the role of sterols in the biological membrane under a variety of experimental conditions.