In a recent Talking Point article1 in TiBS, Hofmann and Dixit raise several complex issues. We welcome the initiation of a serious discussion of this subject. However, we believe that, in their discussion of several of these issues, the authors either fail to include all pertinent data or misinterpret existing data. Is there agonist-induced sphingomyelin hydrolysis and ceramide generation? Hofmann and Dixit question the existence of agonist-induced changes in sphingolipid levels. They specifically challenge the use of the diglyceride kinase (DGK) assay for quantitation of ceramide, basing their argument on a publication by Watts and colleagues2. Watts et al. 2 reported that, when the DGK assay registered an increase in ceramide levels following cell stimulation, mass spectrometry failed to do so. Although this might have resulted from misuse of the DGK assay, and therefore spurious detection of ceramide (see below), note that the documentation of agonist-induced ceramide elevation is based on a solid set of observations.(1) Approximately half of the published studies on ceramide measured elevation in ceramide levels by benzoylation, charring, O-phthalaldehyde derivatization or various radiolabeling methods, demonstrating responses consistent with observations made by the DGK assay. These changes have been detected in many tissues and cell types in response to tumor necrosis factor (TNF), FAS, heat, IgM, interleukin 1 (IL-1), chemotherapeutic agents and many other inducers of stress.(2) As in the case of any other assay, the DGK assay must be used under appropriate conditions to obtain reliable results (see letter by Perry and Hannun3 in this issue). Moreover, the use of the DGK assay in assessing cellular ceramide levels has been validated by several studies that have measured ceramide by more than one method4–6.(3) Several well-controlled studies have coupled elevations in ceramide levels with quantitative decreases in the levels of its precursor, sphingomyelin (SM), usually measured by radiolabeling techniques or phospholipid-phosphorus measurement7, 8. In addition, recent studies confirm these observations by electrospray mass spectrometry. Normal-phase HPLC with on-line electrospray mass spectrometry reveals that, within 30 min of activation of FAS, as much as 40% of total sphingomyelin in HEY and Caov-3 ovarian cancer cells, and in primary cultures of ovarian thecal cells, is hydrolysed9, 10. In addition, numerous studies show concomitant activation of neutral and/or acidic sphingomyelinases (SMases). Given that SMases are the only enzymes known to act on SM in mammalian cells, and that