Pneumolysin (PLY), an important protein virulence factor of the human bacterial pathogen Streptococcus pneumoniae, could be a candidate for inclusion in a new anti-streptococcal vaccine. PLY solution species from monomer via multimeric intermediates to ring-shaped oligomers were studied with time-dependent sedimentation velocity in the analytical ultracentrifuge (AUC). Hydrodynamic bead modeling was used to interpret the data obtained. PLY remained mostly monomeric in solution; intermediate PLY multimers were detected in small quantities. Current understanding of PLY molecular mechanism is guided by a model built on the basis of its homology with perfringolysin O (PFO) for which there is an atomic structure. PFO, a virulence factor of the organism Clostridium perfringens, has almost the same molecular mass as PLY and shares 48% sequence identity and 60% sequence similarity with PLY. We report a comparative low-resolution structural study of PLY and PFO using AUC and small-angle x-ray scattering (SAXS). AUC data demonstrate that both proteins in solution are mostly monodisperse but PLY is a monomer whereas PFO is mostly dimeric. Ab initio dummy atom and dummy residue models for PFO and PLY were restored from the distance distribution function derived from experimental small-angle x-ray scattering curves. In solution, PLY is elongated, consistent with the shape predicted by its high-resolution homology model. The PFO dimer is also an elongated particle whose shape and volume are consistent with a staggered antiparallel dimer.