Abstract Reduction of NO − 2 by the Cu-containing nitrite reductase from Achromobacter cycloclastes produces NO as the primary product initially, but as NO accumulates, NO production levels-off and N 2O production becomes significant. Reaction of the enzyme with NO − 2 in the presence of NO increases the amount of N 2O product significantly, while trapping the NO product as nitrosylhemoglobin or rapid, removal of NO by sparging results in no detectable N 2O production. Reaction of the enzyme with 15NO − 2 in the presence of 14NO results in rapid formation of the mixed isotope product ( 14N, 15N)O in ca. 45% yield. In contrast, the presence or absence of NO has no effect on N 2O production by a prototypical heme cd 1-containing nitrite reductase. These results are consistent with formation of a labile Cu +−NO + species in the copper enzyme, which normally decomposes to NO. Production of N 2O requires that the released NO must rebind to the enzyme to combine with a second NO − 2 or a species derived therefrom.