Yeast sphingolipids can be metabolically labeled with exogenously added radioactive precursors. Here we describe a method to label ceramides and complex sphingolipids of Saccharomyces cerevisiae with a radioactive ceramide precursor, D-erythro-[4, 5-³H]dihydrosphingosine. This protocol is used to study the biosynthesis, transport and metabolism of sphingolipids in yeast.