Pneumolysin (PLY), an important virulence factor of Streptococcus pneumoniae, is one of the members of thiol-activated cytolysins (TACYs) consisting of four domains. TACYs commonly bind to membrane cholesterol and oligomerize to form transmembrane pore. We have constructed full-length and various truncated PLYs to study the role of domains of PLY in the cytolytic activity. Full-length PLY had binding ability to both cell membrane and immobilized cholesterol. A truncated PLY which comprised only domain 4 molecule, the C-terminal domain of PLY, sustained the binding ability to cell membrane and cholesterol, whereas domain 1-3 molecule had no binding ability to them. Furthermore, the domain 4 molecule inhibited both the membrane binding and the hemolytic activity of full-length PLY. Accordingly, the present results provided the direct evidence that domain 4 was essential for the initial binding to membrane cholesterol and the interaction led to the subsequent membrane damage process.