Negative-ion electrospray ionization tandem quadrupole mass spectrometry provides a useful method for the structural characterization of ceramides. Fragment ions referring to the identities of the fatty acid substituent and of the long chain base of the molecules are readily available and the structure of ceramides can be easily determined. A unique fragmentation pathway which leads to formation of the fatty acid carboxylate anions (RCO− 2) was observed. This fragmentation is initiated by cleavage of the C2-C3 bond of the LCB to yield a N-acylaminoethanol anion ([RCONHCH 2 CH 2 O]−), followed by rearrangement to a carboxyethylamine ([RCO 2 CH 2 CH 2 NH]−) intermediate, which further dissociates to a RCO+ 2 ion. This pathway is confirmed by the CAD tandem mass spectrum of the synthetic N-acylaminoethanol standard and of the deuterated analogs of ceramides obtained by HD exchange. The observation of RCO− 2 ion species permits an unambiguous identification of the fatty acyl moiety of ceramides. Tandem mass spectrometry methods for characterization of structural isomers of ceramides using product-ion scanning and for identification of specific ceramide subclasses in biological mixtures using neutral loss scanning are also demonstrated.