This study investigated whether cell size correlates with phenotype and proliferative capacity in human corneal epithelial cells. Primary cultured human corneal epithelial cells were sorted by flow cytometry based on forward scatter profile in comparison with the profile of beads of known size. Four fractions (A, B, C, and D) of cells ranging in size from 10 to 16, 17 to 23, 24 to 30, and >or=31 microm in diameter, respectively, were collected to evaluate their 5-bromo-2-deoxyuridine (BrdU) label retention properties, cell phenotype, and clonal growth capacity on a 3T3 fibroblast feeder layer. Among these four populations, cell size was shown to positively correlate with the expression of the differentiation markers keratin (K) 3, K12, and involucrin and inversely with the levels of stem cell-associated markers DeltaNp63 and ABCG2 and with colony-forming efficiency (CFE) and growth capacity. Population A with the smallest size, accounting for 11.0%+/-4.5% of the entire population, contained the greatest number of BrdU label-retaining slow-cycling cells, displayed the highest percentage of cells immunopositive to p63 and ABCG2 and negative to K3 and involucrin, expressed the highest levels of DeltaNp63 and ABCG2 mRNA and the lowest levels of K3, K12, and involucrin, and possessed the highest CFE and growth capacity. These findings suggest that cell size correlates with cell differentiation phenotypes and proliferative capacity in human corneal epithelial cells. The smallest cells in population A seem to be enriched for putative stem cells, and small cell size may represent one of the important properties of adult corneal epithelial stem cells.