BACKGROUND/AIMS: Topical mitomycin C (MMC) therapy has been used for treatment of ocular surface squamous neoplasia (OSSN) since 1994. Relatively few studies have reported the cellular changes in ocular surface following MMC. METHODS: Impression cytology was studied in four patients with ocular surface squamous neoplasia, either primary or recurrence after previous excisional biopsy. The authors studied samples obtained using Millipore filters at intervals between 4 and 17 weeks after commencement of MMC, and compared them with pretreatment cytology. RESULTS: MMC induced changes of cytomegaly, cytoplasmic vacuolation, nucleomegaly with nuclear wrinkling, and binucleation or multinucleation were seen in some cells in all samples. However, nuclear/cytoplasmic (N/C) ratio in these enlarged cells was normal. These changes mimicked those seen following radiation therapy in uterine cervix. Changes of increased nuclear and cell size with increased N/C ratio were seen in some dysplastic cells. The predominant form of cell death was apoptosis with fewer cells showing necrosis. CONCLUSIONS: MMC appears to produce cell death in OSSN by apoptosis and necrosis. Cellular changes related to MMC mimic those caused by radiation-cytomegaly, nucleomegaly, and vacuolation. MMC related changes may persist in ocular surface epithelium for at least 8 months following MMC therapy.