The mass spectra of the methyl esters of twenty-one metabolically important N-acylglycines are reported. The spectra are discussed in terms of their fragmentations and their application to studies of metabolic diseases. In particular, the high relative abundance of the peak at m/e 30 in the spectra of many aliphatic acylglycine methyl esters was studied by using ([2,2-2H2]propionyl)glycine and high resolution mass spectrometry. This fragment was assigned to the even-electron ion [CH2 = NH2]+.