It is generally believed that plasma membrane glycoconjugates influence corneal epithelial cell migration after wounding. Previous studies have focused on the role of glycoproteins in this event. The present study was designed to determine whether migration-specific glycolipids are synthesized by epithelium of healing rabbit corneas. Migrating and nonmigrating rabbit corneal epithelia were incubated with [3H]-galactose in an organ culture system for 48 hr. At the end of the labeling period, a neutral glycosphingolipid (NGSL) fraction was isolated from each radiolabeled epithelium and was analyzed by thin-layer chromatography. Three radiolabeled NGSL components, M1, M2 and M3 (M1-M3), were present in significantly higher amounts in the extracts of migrating as compared to nonmigrating epithelium. Chromatographic mobility of M3 was similar to that of a standard glucosylceramide; M1 and M2 migrated more slowly than M3. For characterization of the migration-related NGSL, a large amount of the starting material is required. Experiments, therefore, were conducted using cell cultures of rabbit corneal epithelium. Confluent (nonmigrating) cell cultures of rabbit corneal epithelium were found to synthesize either minimal or undetectable amounts of NGSL M1-M3. In contrast, we found that the NGSL M1-M3 are synthesized as major components by sparse (migrating) corneal epithelial cell cultures. Components M1-M3 were synthesized as major components by sparse cultures even in the absence of cell mitosis. This suggests that the increased synthesis of components M1-M3 by sparse cell cultures may be related to cell migration rather than cell mitosis.(ABSTRACT TRUNCATED AT 250 WORDS)