A new, simple method for isolating retinal pigment epithelial (RPE) cells from rat eyeballs was developed: incubation of the eyeballs in 0.1% proteinase K solution. This method yielded an average of 4 x 10(4) viable RPE cells from each Sprague-Dawley rat eyeball in 1 h. These RPE cells were hexagonal and had cytokeratin in their cytoplasm and numerous microvilli on their surface, similar to RPE cells evaluated in vivo. The results of this study thus show that our method for incubating eyeballs in proteinase K can provide good-quality RPE cells in sufficient quantities for study in a short time.