Cell migration is a fundamental process of wound repair in biological systems. In an attempt to identify plasma membrane glycoconjugates which mediate cell migration, migrating and nonmigrating rabbit corneal epithelia were analyzed for reactivity with monoclonal antibodies (mAbs) specific for unsubstituted N-acetyl-lactosamine (mAb 1B2), Le(x) (mAbs 7A and MMA), and sialyl Le(x) (mAb CSLEX1) carbohydrate chains of neolactoglycoconjugates. Immunohistochemical analysis indicated that regardless of whether the epithelia analyzed were from corneas of animals in vivo, corneas in organ culture, or cells in tissue culture, migrating cells stained intensely with mAb 1B2, whereas nonmigrating cells either did not stain or stained only weakly. mAbs MMA and 7A stained migrating epithelium as well as basal and middle cell layers of normal, nonmigrating epithelium. mAb CSLEX1 did not stain wounded corneas but stained the superficial cell layer of normal corneal epithelium. Biochemical analyses by TLC immunostaining revealed the presence of three mAb 1B2-reactive glycosphingolipids (GSL), neolactotetraosyl-(nLc4, paragloboside), neolactohexaosyl- (nLc6), and neolacto-octaosylceramide (nLc8) in migrating epithelia. In contrast, nonmigrating epithelia contained only trace amounts of these glycolipids. Exogenous addition of nLc4, but not various other GSLs including a Le(x)-GSL (SSEA-1), stimulated re-epithelialization of wounds in an experimental model of corneal epithelial wound healing. Moreover, re-epithelialization of wounds was significantly inhibited by mAb 1B2 but not by mAb MMA. The data suggest that neolacto-GSLs of corneal epithelium may be among the molecules which mediate healing of corneal epithelial wounds by influencing cell migration.