Streptococcus pneumoniae is the causative agent of multiple diseases, including otitis media, pneumonia, bacteremia, and meningitis. Pneumolysin (Ply), a member of the cholesterol-dependent cytolytic pore-forming toxins, is produced by virtually all clinical isolates of S. pneumoniae, and strains in which the Ply gene has been deleted are severely attenuated in mouse models of infection. In contrast to all other members of the cholesterol-dependent cytolysin family, Ply lacks a signal peptide for export. Instead, Ply has been hypothesized to be released upon autolysis or, alternatively, via a nonautolytic mechanism that remains ill defined. We determined by use of cell fractionation and Western blotting that, during in vitro growth, exported Ply is localized primarily to the cell wall compartment in 18 different serotypes in the absence of detectable cell lysis. Hemolytic assays revealed that this cell wall-localized Ply is active. Additionally, cell wall-localized Ply is accessible to extracellular protease and is detergent releasable.