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C2-ceramide (N-acetyl-sphingenine) is often used as an analog to study ceramide-mediated cellular processes. According to Lee et al. [J. Biol. Chem. 271 (1996), 209–217], C2-ceramide is formed by an acetyl transfer from platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) to sphingenine. To substantiate these unconfirmed findings, we (i) developed a method to quantify C2-ceramide and (ii) analyzed C2-ceramide levels in Pex5−/- mice, a model for Zellweger syndrome, in which the synthesis of ether lipids such as PAF is impaired. The presence of C2-ceramide could be established in brain (±10 pmol/g) and liver (±25 pmol/g) from control mice, and was approximately 5000-fold less than the main long-chain ceramide species. In Pex5−/- mice, C2-ceramide levels did not differ significantly compared to control tissues. Given the presence of a ceramide kinase in mammals, phosphorylation of C2-ceramide by human ceramide kinase (HsCERK) was tested. C2-ceramide appears to be a good substrate when albumin is used as carrier. In CHO cells overexpressing HsCERK, phosphorylation of exogenously added C2-ceramide could also be demonstrated. Our data indicate that C2-ceramide is present in mammalian tissues and can be converted to C2-ceramide-1-phosphate, in addition to other documented metabolic alterations, but does not seem to be linked to ether lipid metabolism.