Abstract Optical absorption, mcd, and epr spectroscopy have been used to characterize the azide and imidazole derivatives of oxidized Pseudomonas nitrite reductase. At pH 7.0 azide binds solely to heme d 1 with an affinity constant, K aff = 360 M −1, whereas imidazole binds to both hemes c and d 1 with k aff = 35 and 55 M −1, respectively. Low-temperature mcd and epr spectroscopy indicate that c and d 1 are low-spin ferrihemes in both derivatives, although the epr of the heme d 1-azide component is very weak and requires explanation. Attempts to obtain a high-spin heme d 1 in the intact enzyme using the weak field ligands fluoride and thiocyanate have proved unsuccessful. Electron paramagnetic resonance experiments involving an oxidized enzyme derivative in which heme d 1 is complexed by NO, and hence epr silent, have enabled unambiguous assignment of the epr spectrum of Pseudomonas nitrite reductase.