The functional significance of p63 in regulating cell proliferation in various stratified epithelial cells has previously been proposed. More than six isoforms have been reported for this protein; however, it is not yet clearly understood how functionally different these isoforms are. To investigate how these isoforms are used in ocular surface epithelia, we studied the spatial distribution of p63 isoforms within human ocular surface epithelia. Individual layers (basal, intermediate, and superficial) of the human ocular surface epithelia (cornea, limbus, and conjunctiva) were selectively obtained using a laser micro-dissection device. These samples were equally amplified and subjected to RT-PCR analysis with primer pairs, which specifically amplify each of five isoform-determining regions or each of six p63 isoforms. Regarding the N-terminal region, the TA domain was not detected in all samples, while a DeltaNp63 specific region was detected in the basal-intermediate region of all types of epithelia and in the superficial layer of the limbus. Regarding the C-terminal region, an alpha-isoform specific region was detected in all layers of the conjunctiva and limbus, as well as in the basal to intermediate layers of the cornea. A beta-isoform specific region was detected in the basal to intermediate layers of the limbus. A gamma-isoform specific region was detected in almost all layers of all epithelia. Among the six p63 isoforms, only DeltaNp63alpha was detected in the basal to intermediate layers of the limbus and conjunctiva. These results suggest that DeltaNp63alpha is the most dominant isoform within human ocular surface epithelia. This isoform may contribute, at least in part, to the maintenance of cell proliferative capacity within the ocular surface epithelia.