Solid-phase extraction (SPE) methods are easy, rapid, and reliable. Their growing popularity is in part due to their operational simplicity and cost reduction in solvents, and partly because they are easier to automate. Sphingolipids are implicated in various cellular events such as growth, differentiation, and apoptosis. However, their separation by small SPE cartridges has attracted limited attention. Here we describe an SPE procedure on aminopropyl cartridges that by sequential elution allows the separation of a lipid mixture into free ceramides, neutral glycosphingolipids, neutral phospholipids (sphingomyelin), and a fraction containing the acidic phospholipids and phosphorylated sphingoid bases, phosphoceramides and sulfatides. Individual components are obtained in high yield and purity. We applied the procedure to obtain data on separation of [(3)H]myristic acid-labeled sphingolipids from fish gills, and from human melanoma tumor tissue. Individual lipids in the SPE fractions were identified by chromatography on several high-performance thin-layer chromatography (HPTLC) systems. The chromatographic behavior of free sphingoid bases is also reported.