Previously, we reported that a salicylate 1-monooxygenase from Pseudomonas sp. ATCC 29352 biotransformed CL-20 (2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaaza-isowurtzitane) (C6H6N12O12) and produced a key metabolite with mol. wt. 346 Da corresponding to an empirical formula of C6H6N10O8 which spontaneously decomposed in aqueous medium to produce N2O, Click to view the MathML source, and HCOOH [Appl. Environ. Microbiol. (2004)]. In the present study, we found that nitroreductase from Escherichia coli catalyzed a one-electron transfer to CL-20 to form a radical anion (CL-20radical dot-) which upon initial N-denitration also produced metabolite C6H6N10O8. The latter was tentatively identified as 1,4,5,8-tetranitro-1,3a,4,4a,5,7a,8,8a-octahydro-diimidazo[4,5-b:4?,5?-e]pyrazine [IUPAC] which decomposed spontaneously in water to produce glyoxal (OHCsingle bondCHO) and formic acid (HCOOH). The rates of CL-20 biotransformation under anaerobic and aerobic conditions were 3.4 � 0.2 and 0.25 � 0.01 nmol min-1 mg of protein-1, respectively. The product stoichiometry showed that each reacted CL-20 molecule produced about 1.8 nitrite ions, 3.3 molecules of nitrous oxide, 1.6 molecules of formic acid, 1.0 molecule of glyoxal, and 1.3 ammonium ions. Carbon and nitrogen products gave mass-balances of 60% and 81%, respectively. A comparative study between native-, deflavo-, and reconstituted-nitroreductase showed that FMN-site was possibly involved in the biotransformation of CL-20.