A sensitive and specific electron capture-gas chromatographic assay was developed for the determination of 7-iodo-1,3-dihydro-1-methyl-5-(2′-fluorophenyl)-2H-1,4-benzodiazepin-2-one (I) and its major metabolites in blood and urine. The overall recovery of I and its N-desmethyl metabolite (II) from blood is apparently quantitative. The recovery of the major urinary metabolite, the N-desmethyl-3-hydroxy analog (IV), and the minor metabolites, the N-desmethyl analog (II) and the N-methyl-3-hydroxy analog (III) added to urine as authentic reference standards ranged from 80 to 85%. The sensitivity limits of detection are of the order of 2-3 ng of I and 4-5 ng of II per ml of blood or urine. The method was applied to the determination of blood levels and the urinary excretion pattern in a dog following oral and intravenous administration of a 1-mg/kg dose (total 13 mg), and in man following the intravenous administration of single 5- and 10-mg doses. The N-desmethyl metabolite II was more predominant in dog blood than was the orally or intravenously administered I, but II was barely measurable in human blood.