This paper is the first report on the use of the electron microscopy autoradiography technique to detect metabolically tritium labeled sphingolipids in intact cells in culture. To label cell sphingolipids, human fibroblasts in culture were fed by a 24 hours pulse, repeated 5 times, of 3 x 10(-7) M [1-(3)H]sphingosine. [1-(3)H]sphingosine was efficently taken up by the cells and very rapidly used for the biosynthesis of complex sphingolipids, including neutral glycolipids, gangliosides, ceramide and sphingomyelin. The treatment with [1-(3)H]sphingosine did not induce any morphological alteration of cell structures, and well preserved cells, plasma membranes, and intracellular organelles could be observed by microscopy. Ultrathin sections from metabolic radiolabeled cells were coated with autoradiographic emulsion. One to four weeks of exposition resulted in pictures where the location of radioactive sphingolipids was evidenced by the characteristic appearance of silver grains as irregular coiled ribbons of metallic silver. Radioactive sphingolipids were found at the level of the plasma membranes, on the endoplasmic reticulum and inside of cytoplasmic vesicles. Thus, electron microscopy autoradiography is a very useful technique to study sphingolipid-enriched membrane domain organization and biosynthesis.