Cytochrome b(558) purified from pig neutrophils was studied to characterize the spin state of the heme iron in relation to its O-2-radical-anion generating activity. ESR spectra of cytochrome b(558) either from resting or stimulated neutrophils showed a low-spin hemoprotein with g(1,2,3) of 3.2, 2.1, and 1.3 (estimated), At physiological pH, the oxidized cytochrome b(558) is in a purely low-spin state, On lowering or raising pH from 7, the spin state changes to high-spin. The ESR spectrum of high-spin cytochrome b(558) was identical to that of methemoglobin, suggesting that the axial-ligand type in both hemoproteins may be the same, i.e. histidine is the fifth ligand. The ratio of the low-spin to high-spin heme in cytochrome b(558) was evaluated by magnetic circular dichroism spectroscopy. The pH of cytochrome b(558) was varied to form different ratios of the low-spin to high-spin states of the heme, and its O-2-radical-anion generating activity was examined in cell-free systems, O-2-radical-anion farming activity decreased concomitant with loss of the low-spin heme, which provides direct evidence that the low-spin state of cytochrome b(558) is essential to generate O-2-radical-anion and the heme retains the low-spin state through the redox cycle.