Vitamin A is known to regulate the proliferation and differentiation of corneal epithelial cells and preserved conjunctival goblet cells and has been used in the treatment of disease of the eye such as dry eye and superior limbic keratocon-junctivitis for some time. This study was undertaken in order to evaluate the efficacy of retinol palmitate aqueous ophthalmic solution under development for the treatment of dry eye failing to respond to the conventional therapy with artificial tears or cornea-protective drugs. Retinol palmitate ophthalmic solution was applied repeatedly for 4 consecutive weeks. Before and after instillation therapy, brush cytology (Cytobrush-S®) was performed and cytodiagnosis was made for keratinized cells, nonkeratinized cells, goblet cells and inflammatory cells on samples prepared using an automated smear apparatus (ThinPrep®). In dry eye, an increase in goblet cells (1.3 ± 2.6→2.1 ± 1.8 cells/slides), a decrease in keratinized cells (11.2 ± 16.5→5.2 ± 10.9 cells/300 cells) and, hence, an increase in nonkeratinized cells (287.3 ± 16.6→293.4 ± 11.4/300 cells) were found after treatment with retinol palmitate. As to inflammatory cells, there was no change from the pretreatment baseline (1.4 ± 1.4→1.4 ± 1.3 cells/300 cells). These results demonstrate that brush cytology suggests the efficacy of retinol palmitate ophthalmic solution in dry eye treatment.