Galactosylceramide (GalCer) and cerebroside sulfate (CBS) are the major glycolipids found in myelin. They occur in greater concentrations in this membrane than any other. Recently, it was reported that these two glycolipids can participate in a heterotypic carbohydrate-carbohydrate interaction [Hakomori et al. (1991) Glycoconjugate J. 8, 178]. In the present study, the effect of changes in the ceramide composition of both GalCer and CBS on this interaction has been examined. The interaction was monitored by measuring the aggregation of small unilamellar phosphatidylcholine/cholesterol liposomes containing GalCer with similar liposomes containing CBS, through the increase in optical density at 450 nm. Aggregation depends on the addition of a divalent cation and varies inversely with the ionic radius of the cation. Aggregation occurred at millimolar concentrations of divalent cation and was inhibited and reversed by the addition of EDTA. A lesser degree of homotypic self-aggregation of GalCer and of CBS liposomes also occurred in the presence of divalent cations, but the sum of this self-aggregation was significantly less than the heterotypic interaction between the two types of liposomes. Changes in the ceramide composition of GalCer and CBS significantly affected the extent of their interaction with each other. Increasing the fatty acid chain length of either GalCer or CBS resulted in increased aggregation. Hydroxylation of the fatty acid also increased the degree of aggregation of GalCer and CBS liposomes. These findings indicate that a divalent cation-mediated GalCer-CBS interaction could play a role in cell recognition and membrane adhesion phenomena such as the formation of compact multilamellar myelin.(ABSTRACT TRUNCATED AT 250 WORDS)