Hydroxylamine oxidoreductase (HAO) from Nitrosomonas europaea catalyzes the oxidation of NH2OH to NO2-The enzyme contains eight hemes per subunit (63 kDa) which participate in catalytic function and electron transport. In the resting ferric state of the enzyme, we find that these hemes can be categorized on the basis of quantitative integer-spin and multifrequency EPR data as follows: (a) Four hemes are noninteracting, ferric c-type hemes, (b) Two hemes are low-spin and weakly spin-coupled through an interaction which is best described as dipolar on the basis of frequency-dependent g values, (c) The final two hemes are exchange-coupled; this cluster is the origin of a new integer-spin EPR signal which can be quantitatively associated with the reactive P460heme of HAO. Accordingly, treatments that affect the optical properties of P460 and catalysis of the enzyme, such as substrates, CN-, or H2>O2, all caused the integer-spin resonance to vanish. For reduced HAO, a second integer-spin EPR signal is observed which derives from one high-spin ferrous heme; previous Mossbauer studies have shown that the only such heme is P460. An analysis of potential electronic models for the exchange-coupled cluster indicated that the interacting spins are both S = ½. It thus appears that the active site of HAO contains a pair of low-spin hemes which are coupled in a manner not previously observed in proteins. The structure of the diheme cluster and in the presence of substrates or inhibitors, and its bearing on catalysis by HAO is discussed.