Nitrite reductase as isolated from Desulfovibrio desulfuricans shows a complex set of rhombically distorted high-spin ferric heme and low-spin ferric heme resonances at 11 oK. These resonances disappear (due to reduction to the diamagnetic ferrous state) on enzymatic reduction with hydrogen, hydrogenase from D. vulgaris and FAD as redox mediator. The addition of nitrite to the reduced enzyme results in reoxidation of nitrite reductase as evidenced by the reappearance of most of the initial signal intensities of high-spin and low-spin ferric heme resonances. Simultaneously an intense and unusual broadened signal appears in the g=2 region, suggesting the formation of a novel heme-nitric oxide signal with the main g-value at 2.08. Confirmation of this heme-nitric oxide complex was demonstrated by reoxidation of reduced enzyme with 15NO 2 −1 instead of 14NO 2 −1 resulting in a decrease from three to two of the hyperfine interaction pattern of nitrogen. Thus nitrite reduction to ammonia by nitrite reductase occurs via a heme-nitric oxide intermediate as reported with spinach nitrite reductase.