Ceramides are intermediates in the biosynthesis of membrane sphingolipids. These biomolecules are also important as second messengers in signal transduction pathways controlling cell growth. We have developed two reversed-phase high pressure liquid chromatography (RPHPLC) techniques for identification and quantification of ceramides from mammalian cells. One method was based on atmospheric pressure chemical ionization-mass spectrometry (APCI-MS) detection of ceramides and had the advantage of requiring minimal sample preparation, yielding significant structural information, and affording high sensitivity. The second method relied on perbenzoylation of the ceramides and detection at 230 nm. The predominant ceramides detected in the human leukemic HL-60 cell were N-(palmitoyl)-sphingosine, N-(nervonyl)-sphingosine, and N-(lignoceroyl)-sphingosine. When selected ion monitoring was used with RPHPLC/APCI-MS, approximately 2.2 pmol N-(palmitoyl)-sphingosine and 1.7 pmol N-(nervonyl)-sphingosine were observed in an extract from 40,000 HL-60 cells. Perbenzoylation with benzoyl chloride permitted RPHPLC separation and 230 nm UV absorbance detection of the trisbenzoyl derivatives of sphingosine, N-(palmitoyl)-sphingosine, N-(nervonyl)-sphingosine, and N-(lignoceroyl)-sphingosine in the HL-60 cells. These results demonstrate the utility of utilizing two different methods coupled with APCI-MS for the quantification and identification of ceramides in biological samples.