The methane negative chemical ionization (NCI) mass spectra of the medically important 1,3-dihydro-5-phenyl-1,4-benzodiazepin-2-ones generally consisted solely of M- and (M-H)- ions. Attempts to find the location of the H lost in the generation of the (M-H)- ion were unsuccessful, although many possibilities were eliminated. A Hammett correlation analysis of the relative sensitivities of a series of 7-substituted benzodiazepines suggested that the initial ionization takes place at the 4,5-imine bond. For certain benzodiazepines, the (M-H)- ion generated by methane NCI was 20 times more intense than the MH+ ion generated by methane positive chemical ionization (PCI). By using NCI, a sensitive and simple GC-MS assay for nordiazepam was developed that can quantitate this important metabolite of many of the clinically used benzodiazepines in the blood and brain of rats.