Phenol-soluble modulin alpha 3 (PSM alpha 3) is a cytotoxic peptide secreted by virulent strains of Staphylococcus aureus. We used a stereochemical strategy to examine the mechanism of PSM alpha 3-mediated toxicity. One hypothesis is that PSM alpha 3 toxicity requires fibril formation; an alternative is that toxicity is caused by soluble forms of PSM alpha 3, possibly oligomeric. We find that the unnatural enantiomer (D residues) displays cytotoxicity comparable to that of L-PSM alpha 3. Racemic PSM alpha 3 is similarly toxic to enantiopure PSM alpha 3 (L or D) under some conditions, but the toxicity is lost under conditions that cause racemic PSM alpha 3 to aggregate. A crystal structure of racemic PSM alpha 3-NH2 displays an alpha-helical secondary structure and a packing pattern that is reminiscent of the cross-alpha arrangement recently discovered in crystals of L-PSM alpha 3. Our data suggest that the cytotoxicity of PSM alpha 3 does not depend on stereospecific engagement of a target protein or other chiral macromolecule, an observation that supports a mechanism based on membrane disruption. In addition, our data support the hypothesis that toxicity is exerted by a soluble form rather than an insoluble fibrillar form.