PURPOSE: Tandem scanning confocal microscopy was performed on two patients with Acanthamoeba keratitis to provide images detailing characteristic findings of the disease. Although tandem scanning confocal microscopy of Acanthamoeba has been described in previous reports, Acanthamoeba keratitis has not been fully characterized with this instrument. In vivo confocal micrographs showed the double-walled structure of the Acanthamoeba cyst and associated radial keratoneuritis (perineuritis). METHODS: We reviewed the records of two patients with a clinical diagnosis of Acanthamoeba keratitis, one with culture-proven Acanthamoeba and the other with a suspected Acanthamoeba infection. Slit-lamp biomicroscopy and in vivo tandem scanning confocal microscopy were performed. The images obtained were compared with images from patients without corneal disease. RESULTS: High-contrast round bodies suggestive of Acanthamoeba cysts, as previously described, and irregular forms suggestive of Acanthamoeba trophozoites were found by tandem scanning confocal microscopy. Additionally, we showed conclusively that under certain circumstances (that is, corneal scarring) tandem scanning confocal microscopy can resolve the double-walled structure of the Acanthamoeba ectocyst surrounding the endocyst. Furthermore, radial keratoneuritis was demonstrated, consisting of an irregularly swollen nerve fiber with probable amoebic infiltration. CONCLUSIONS: Confocal microscopy can be a useful, noninvasive imaging technique helpful in the study, diagnosis, and treatment of Acanthamoeba keratitis.