Pseudomonas aeruginosa is a common cause of corneal infections, particularly among users of soft contact lenses. Previous studies with chemically induced mutants deficient in alkaline protease (AP) or elastase (LasB) suggested that these proteases contributed to the rapid liquifactive stromal necrosis characteristic of P. aeruginosa corneal infections. Because these mutants might harbor other chromosomal changes that could affect virulence, the role of these proteases in the pathogenesis of corneal disease (as well as a second elastase, LasA protease) was reexamined by constructing isogenic mutants deficient only in these enzymes. Allelic exchange was used to construct mutants of P. aeruginosa PAO1-V deficient in AP (PAO1-V AP[ - ]), LasB and LasA protease (PDO801 LasB[ - ]), or all three proteases (PDO801 TM). These mutants were then evaluated for virulence using mouse scratch and rabbit intrastromal injection models of corneal disease. Loss of AP significantly increased disease scores in the rabbit (P < 0.030) but not the mouse (P > 0.060) model of infection. Loss of both elastases had no effect on ocular virulence in either animal model of corneal disease (P > 0.100). The loss of all three proteases significantly decreased disease scores in the rabbit (P < 0.035), but not in the mouse (P > 0.110). Taken together, these data suggest that AP, LasB, and LasA protease are not essential for initiating or maintaining a corneal infection. Furthermore, AP appears to be an important mediator of pathology depending on the location of the organism within the cornea and whether or not concomitant elastolytic activity is present.