The stem cells and transient amplifying cells of the corneal epithelium are thought to be localized in the limbal and corneal basal epithelium, respectively. To study the differential regulation of proliferation of these progenitor cells, a defined, serum-free, clonal growth assay was developed for central (CC) and peripheral (PC) corneal and limbal (L) epithelial cells. After incubation in Dispase II (1.2 U/ml; 1 hr for PC and CC and 3 hr for L) and subsequent brief trypsin-ethylenediaminetetraacetic acid digestion, 18 or 180 single cells/cm2 were seeded in MCDB 151 medium supplemented with insulin, transferrin, selenium, hydrocortisone, epidermal growth factor (EGF), phosphoethanolamine, ethanolamine, and calcium. During the first week of culture, the cells gradually developed an increasing number of colonies, and the mean colony-forming efficiency on day 6 for L was 4.2 +/- 2.4%, significantly lower than 11.4 +/- 5.9% for PC and 12.8 +/- 7.6% for CC (P less than 0.003). Colony morphology was identical in L, PC, and CC with small, elongated cells more cohesive in the center but more migratory in the periphery. There were no differences in immunofluorescent staining with monoclonal antibody AE-5, indicating the corneal derivation of all colonies. Cultures could be passaged on day 14 and grown for more than 3 weeks with increasing desquamation. Addition of a mixture of trace metals to yield MCDB 153 did not enhance growth; increased selenium concentrations were inhibitory. Elimination of EGF from the supplement abolished most of the clonal growth. The lower rate of L proliferation might be explained by the absence of serum and stromal mitogens. This culture system seems preferentially to support transient amplifying cells and allows investigation of the differentiation of isolated corneal stem cells to transient amplifying cells or the proliferation and differentiation of transient amplifying cells by various factors without the interaction of undefined serum components or paracrine influences from other cells.