Human cultured fibroblasts were fed with G,, ganglioside species isotopically labeled at C3 of C1 Ssphingosine, or at C3 of C18-sphinganine, or at the sialic acid acetyl group, and with C18-sphingosine and CIS-sphinganine, both labeled at C1. After a lipid pulse the cells were subjected until 7-day chase; measurements were then made of the radioactive products resulting from the administered long-chain base and ganglioside species catabolism and the salvage processes of catabolic fragments. From the data we drew the following conclusions.The G,, species differing in the long-chain base structure were taken up by the cells and metabolized. About 80% of the total catabolic CIS-sphingosine and C18-sphinganine were recycled for the biosynthesis of complex sphingolipids, the rest being degraded.Results obtained by administering ganglioside species of G,, containing radioactive sphingosine or the free radioactive sphingosine to fibroblasts suggested the existence in the cells of two quite separate pools of sphingosine. One pool was the direct result of either the catabolism of radioactive G,, highdensity microdomains or the diffusion of exogenous sphingosine into the cell; this pool was mainly used for the biosynthesis of the G,, species that contain palmitic and stearic acids. The other pool of sphingosine, the cell basal pool, came from the catabolism of radioactive sphingolipids in the recycling of sphingosine, and was used for the biosynthesis of the G,, species that mainly contain very long fatty acid chains, the main fibroblast endogenous species of GD3. Administration of the ganglioside species of G,, containing sphinganine or free sphinganine to fibroblasts yielded the G,, species containing mainly very long-chain fatty acids and sphingosine.These results show the possible existence of a pool of ganglioside-derived sphingosine, quite separate from the cell basal pool of sphingosine, suggesting that sphingosine derived from sphingolipid catabolism is reduced to sphinganine before entering the sphingolipid biosynthetic pathway.Keywords: ganglioside ; biosynthesis ; sphingosine ; sphinganine ; recycling.Gangliosides, sialic acid containing glycosphingolipids that are normal components of plasma membranes [I], are biosynthesized in the Golgi apparatus and degraded in the lysosomes [2] to fragments that are recycled for biosynthetic purposes [3-61. Among the ganglioside catabolic fragments, sphingosine has attracted scientific interest and increasing attention. It has been shown to act as an intracellular effector through the modulation of protein kinase C and other enzyme activities [7-91. Most of our present information arises from the administration of sphingosine to cells, but little is known about a potential role of a pool of sphingosine originated from ganglioside catabolism in modulating cell function.Catabolic sphingosine is partly recycled, but much of it is degraded [6, ; thus it is difficult to determine to what degree this recycling affects the overall sphingolipid turnover. Moreover, the de novo biosy...