The aim of this study was to determine the pathogenic role of alpha-, beta-, and gamma-toxins in a rabbit model of Staphylococcus aureus keratitis. S. aureus strains 8325-4, Newman, and their isogenic mutants were intrastromally injected into rabbit corneas. Eyes were scored for pathology by slit lamp examination (SLE), histologic examination, and bacterial colony-forming units (CFU) per cornea were determined. Rabbits were immunized against alpha-toxin and subsequently challenged with S. aureus strain 8325-4 or Newman. All strains grew equivalently to approximately 7 log CFU/cornea at 25 h postinfection. SLE scores at 15, 20, and 25 h postinfection revealed that alpha-toxin-producing strains caused greater corneal pathology than strains deficient in alpha-toxin. A beta-toxin-deficient mutant produced significantly less ocular edema than its parent or rescued strains. The gamma-toxin-deficient mutant, relative to its parent strain or genetically rescued strain, had reduced virulence. These results demonstrate that the virulence of S. aureus involves mainly alpha-toxin and to a lesser extent gamma-toxin, with beta-toxin mediating minimal corneal pathology.