As previously reported, alpha9 integrin is expressed between the merged or fused eyelids of mice at birth, and changes in alpha9 localization occur during lid opening. To determine whether alpha9 and/or additional integrin subunits mediate the emergence and temporary fusion of the eyelids, immunofluorescence and confocal microscopy were used to evaluate the localization of various integrin subunits in the developing ocular surface of the mouse. No detectable beta5, beta6, or beta7 integrins were observed on the epithelia of the ocular surface. alpha2, alpha3, alphav, and beta1 integrins were most abundant in the basal cells beginning at 13.5 days post conception and remained primarily localized to the basal cell layers throughout development. beta4 was localized at the basal surface of the epidermal basal cells beginning at 13.5 days post conception but was not found on the corneal epithelial basal cells until after birth. alpha9 and beta8 integrins were present on suprabasal cells of the epidermis at the leading edge of the eyelid before merger and on the epithelial bridge that forms immediately after these tissues merge, suggesting that they play a role in the initial fusion of the epithelial tissues of the eyelid and in stabilizing the epithelial junction. After birth and into adulthood, beta8 was retained within the suprabasal cell layers of the epidermis, whereas alpha9 became localized to the basal cells of the epidermis, the conjunctiva, and the limbus. The lack of co-localization of beta4 with either alpha9 or beta8 in double-labeling studies suggests that alpha9 and beta8 are restricted to the lateral and apical aspects of those cells in which they are expressed. The presence of tenascin-C and laminin-5 at the epithelial junction site suggests that alpha9: tenascin-C and beta4: laminin-5 interactions may play a role in stabilizing the fusion between lids early on but do not appear to be involved in the movement of the lids across the cornea. The data presented identify specific integrins and matrix proteins that are likely to mediate eyelid fusion.