The ultimate step in chloramphenicol (CAM) biosynthesis is a six-electron oxidation of an aryl-amine precursor (NH2-CAM) to the aryl-nitro group of CAM catalyzed by the non-heme diiron cluster-containing oxygenase Cm1I. Upon exposure of the diferrous cluster to O-2, Cm1I forms a long-lived peroxo intermediate, P, which reacts with NH2-CAM to form CAM. Since P is capable of at most a two electron oxidation, the overall reaction must occur in several steps. It is unknown whether P is the oxidant in each step or whether another oxidizing species participates in the reaction. Mass spectrometry product analysis of reactions under O-18(2) show that both oxygen atoms in the nitro function of CAM derive from O-2. However, when the single-turnover reaction between O-18(2)-P and NH2-CAM is carried out in an O-16(2) atmosphere, CAM nitro groups contain both O-18 and O-16, suggesting that P can be reformed during the reaction sequence. Such reformation would require reduction by a pathway intermediate, shown here to be NH(OH)-CAM. Accordingly, the aerobic reaction of NH(OH)-CAM with diferric Cm1I yields P and then CAM without an external reductant. A catalytic cycle is proposed in which NH2-CAM reacts with P to form NH(O.H)-CAM and diferric Cmll. Then the NH(OH)-CAM rereduces the enzyme diiron cluster, allowing P to reform upon O-2 binding, while itself being oxidized to NO-CAM. Finally, the reformed P oxidizes NO-CAM to CAM with incorporation of a second O-2-derived oxygen atom. The complete six-electron oxidation requires only two exogenous electrons and could occur in one active site.