Cytology using the brush technique is readily available and is a rapid means of establishing a presumptive diagnosis of ocular surface changes. However, those techniques have some limitations when obtaining cells from certain localized areas particularly if using the Cytobrush-S. We have described here a new type of brush (Accellon-M), which can collect the local cells using its spherical tip, and evaluated them by comparing with Cytobrush-S. Furthermore, we differentiated epithelial cells from nonepithelial cells that were collected by brushing, and by a combination of brush cytology and flow cytometry using an anti-keratin antibody, AE-3, which reacts with all basic epithelial keratins. Accellon-M could collect the epithelial cells from conjunctiva as effectively as with the Cytobrush-S, and there were no statistical differences between both groups. AE-3, which is reported as the marker of epithelial cells, were detected quantitatively by a combination of brush cytology and flow cytometry techniques. The result of the present study emphasizes that both the Cytobrush-S and the Accellon-M are valuable for conjunctival brush cytology. An additional positive feature of the Accellon-M may be improved cell collection from the conjunctival epithelium especially when the target cells are in a limited area. The combination of the brush cytology and the flow cytometry technique appears to be a useful adjunct as an additional diagnostic or research tool for use in the detection of various antigens in the conjunctival epithelium.