We reported previously that the incorporation of sugars into glycosphingolipids (GSL) is diminished in SW13 cells that lack a vimentin intermediate filament (IF) network (vim-) compared to vim+ cells. To further analyze the nature of this abnormality, we double-labeled cells with 3H-serine and 14C-sugars. There was no difference between vim+ and vim- cells in the incorporation of serine into GSL, although the usual difference in sugar incorporation was observed. This indicated that the defect in vim- cells was not in the incorporation of sugars into ceramide synthesized de novo by acylation of sphinganine (pathway 1). Sugars can also be incorporated into ceramide synthesized from sphingosine that is derived from catabolism of sphingolipids (pathway 2), and into GSL that recycle through the Golgi apparatus from endosomes (pathway 3). The amount of galactose and glucosamine incorporated into GSL in these three pathways was analyzed by the use of two inhibitors of sphingolipid biosynthesis. beta-Chloroalanine inhibits the de novo synthesis of sphinganine (pathway 1), and fumonisin B1 inhibits the acylation of sphinganine and sphingosine (pathways 1 and 2). We were surprised to observe that in both vim+ and vim- cells only 20-40% of sugar incorporation into GSL took place in pathway 1, and 60-80% of sugar incorporation took place in the recycling pathways. Moreover, in contrast to larger GSL, GlcCer was not synthesized in pathway 3. Our observations indicate that vimentin IF facilitate the recycling of GSL and sphingosine, and that the differences between vim+ and vim- cells are predominantly in pathways 2 and 3. Furthermore, although it is generally believed that virtually all GSL are synthesized in the de novo pathway, these data indicate that the recycling pathways predominate in the incorporation of sugars into GSL in SW13 cells.