Candida albicans is a commensal fungus of the normal flora yet causes opportunistic infection following trauma or surgery and during immunosuppression. C. albicans virulence factors include morphogenesis into invasive filaments, adherence to host cells, and secretion of proteases. This study evaluated the role of fungal hyphal extension in experimental C. albicans keratitis using genetically altered yeast strains. Scarified corneas of adult BALB/c mice were topically inoculated with wild-type (SC5314) or 10 transposon-induced mutant strains of C. albicans and monitored for 4 days post inoculation (PI). In vitro growth kinetics and the yeast strains' ability to bud into pseudohyphae or hyphae were also compared. The wild-type human isolate had a high degree of virulence in the murine cornea, and four fungal strains deficient in genes regulating adherence or encoding membrane proteins did not significantly differ from the parental strain (P>0.3). Five yeast strains deficient in genes involved in filamentation resulted in fully or partially attenuated keratomycosis (P<0.0001). The overall growth kinetics of wild-type and mutant strains were similar in rich media (P>0.9), but mutants with deficient morphogenesis had reduced filamentation in vitro. Phenotypic switching from yeasts to filamentous forms facilitates the establishment and progression of experimental corneal disease by C. albicans.