The cholesterol-dependent cytolysins (CDCs) comprise the largest class of bacterial β-barrel pore-forming toxins and are utilized as virulence factors by numerous gram-positive pathogens. CDCs are generally secreted as soluble monomers that bind membrane cholesterol and assemble into oligomeric prepore complexes prior to undergoing dramatic conformational change into a membrane-spanning pore. In the soluble/prepore form, the CDC domain 3 (D3) is composed of a core β-sheet flanked by two α-helical bundles, which unfurl into trans-membrane hairpins upon conversion to the pore. We have identified a previously uninvestigated conserved motif (F/Y-F/Y-Xn-YGR) in the severe bend of the D3 core β-sheet in the CDCs. Saturation alanine mutagenesis of these residues in PFO (F230Y231-Xn-Y273GR) revealed that the substitution of F230 resulted in a minimal (2-fold) loss of pore-forming activity whereas alanine substitution of Y273 and G274 increased the activity about 3-fold or activity was unaffected, respectively. However, alanine substitution of Y231 or R275 resulted in complete loss of pore forming activity. Studies reveal that R275 contributes to driving the prepore to pore conversion. PFO residue Y231 is situated in a hydrophobic pocket in the pore-forming domain of PFO and other CDCs. This arrangement remains stable in both the soluble and pore forms of PFO. The loss of the aromatic residue at this location destabilizes the soluble PFO monomer and leads to the formation of manifold aberrant oligomeric complexes incapable of converting to the pore. Hence, within this conserved motif residues are present that are necessary for maintaining the proper geometry of the monomer-monomer interaction and driving the prepore to pore conversion.