Sphingomyelin (SM) is an abundant phospholipid in cell membranes. However, owing to the lack of appropriate probes, the subcellular distribution of SM remains unclear. In this study, we examined the localization of SM in COS-1 cells (green monkey kidney cells) by using two SM probes, lysenin and equinatoxin-II (EqtII). Both toxins stained SM in the plasma membrane (PM), and the stains were abolished by sphingomyelin synthase 2 (SMS2) knockdown or sphingomyelinase (SMase) treatment. Simultaneous labeling by the two toxins showed that the PM has heterogeneous SM pools: a SM pool stained by only lysenin, a SM pool stained only by EqtII, and a SM pool stained by both toxins. In permeabilized cells, lysenin exclusively stained late endosomes (LEs) among intracellular organelles, whereas EqtII stained recycling endosomes (REs) in addition to LEs. The intracellular SM stains by EqtII were abolished by sphingomyelin synthase 1 (SMS1) knockdown, but not by SMS2 knockdown. These results indicate that lysenin and EqtII label different SM pools and that SMS2 and SMS1 are responsible for the synthesis of SM in the PM and endomembranes, respectively, in COS-1 cells. The use of the two SM-binding probes may provide more insights into various sphingomyelin-mediated processes in different topological domains.