The central mission for the development of the National Institute of Standards and Technology/National Institutes of Health/Environmental Protection Agency Mass Spectral Library is the acquisition of reference gas chromatography-mass spectrometry data for important compounds and their chemical modification products. The addition of reliable reference data of various derivatives of amino acids to The Library, and the study of their behavior under electron ionization conditions may be useful for their identification, structure elucidation and a better understanding of the data obtained when the same derivatives are subjected to other ionization methods. N-Alkyl-N-perfluoroacyl derivatives of amino acids readily produce previously unreported alkylnitrilium cations of composition [HC≡N-alkyl](+). Homologous [HC≡N-aryl](+) cations are typical for corresponding N-aryl analogs. The formation of other ions characteristic for these derivatives involves oxygen rearrangement giving rise to ions [C(n)F(2n+1)-C≡N(+)C(n)H(2n+1)] and [CnF(2n+1)-C≡N(+)-aryl]. The introduction of an N-benzyl substituent in a molecule favors a process producing benzylidene iminium cations. L-Threonine and L-cysteine derivatives exhibit more fragmentation pathways not typical for other α-amino acids; additionally, the N(ω)- amino group in L-lysine directs the dissociation process and provides structural information on the substitution at the amino functions in the molecule.