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Our previous studies of glycosphingolipids (GSLs) of human umbilical vein endothelial cells (HUVECs) established that globoside and ganglioside GM3 are the most abundant GSLs of HUVECs. Both compounds are located intracellularly, as well as on the cell surface. In this study, we demonstrate that the intracellular globoside and GM3 antigens are associated with the vimentin intermediate filaments of the HUVEC cytoskeleton. Immunofluorescence staining of fixed, permeabilized HUVECs showed colocalization of globoside and GM3 with vimentin but not with tubulin or actin. Both GSLs remained associated with intermediate filaments after perinuclear collapse of the filaments induced by colcemid. Indirect evidence that the globoside epitope is present on a GSL is the loss of staining by anti-globoside after methanol fixation and the absence of anti-globoside reactivity with HUVEC proteins on immunoblots. Colocalization of anti-globoside and anti-vimentin was also demonstrated in cryosections of endothelial cells, which indicates that the observed association was not an artifact induced by exposure of cells to detergent or organic solvent. Association of globoside with intermediate filaments was confirmed by immunoelectron microscopy, which demonstrated the presence of antigen along intermediate filaments, as well as on the cell surface and on lipid vesicles. Interferon-gamma decreased the ratio of surface to filamentous globoside staining, but had the opposite effect on GM3 distribution. Less abundant HUVEC GSLs, including Gb3, nLc4, IV2FucnLc4, and IV3NeuAcnLc4, were not detected along filaments. This is the first report of the association of GSLs with intermediate filaments. We suggest that intermediate filaments may play a role in the transport of GSLs.