Monoclonal antibodies highly selective for developmentally regulated antigens present in the cornea (Zak and Linsenmayer, Dev. Biol. 99, 373-381, 1983) have been used to immunohistochemically evaluate differentiation in intact chick corneas cultured on the chorioallantoic membrane (CAM) of host embryos. One antibody is directed against the epithelial cell layer and the other is against the corneal stromal matrix. It has been established that both antigens recognized by the antibodies are expressed de novo in young explanted corneas and that the stromal matrix antigen is a product of the corneal fibroblasts. Thus expression of the antigens can be used as criteria for overt differentiation of the respective cell types. The antibodies have been employed to assess when the corneal epithelial and stromal cells become capable of autonomous differentiation within isolated corneas. To accomplish this, corneas of various ages were explanted with and without adjacent pericorneal tissues. The results indicate that, under the culture conditions employed, corneal stromal differentiation is dependent on the presence of the lens until stage 28 (51/2-6 days of development), which is the time when invasion of the stroma by pericorneal mesenchymal cells is initiated. After stage 28, the stromal matrix antigen was expressed by isolated corneas irrespective of the presence of the lens. Possibly the lens acts by maintaining the integrity of the corneal endothelial monolayer and thus promoting normal migration of pericorneal mesenchymal cells into the primary corneal stroma, where they undergo differentiation. Conversely, differentiation of the corneal epithelium was independent of any pericorneal structure from the earliest stage examined (41/2-5 days of development). It was even independent of overt stromal differentiation, thus suggesting an early and strong determination for this tissue.