Abstract A concentrated solution of pyrocatechase having a pronounced red color showed a sharp electron spin resonance signal at g = 4.28 known to be due to the high spin state of ferric ion. The signal markedly decreased upon the addition of sodium dithionite and was restored when the solution was exposed to air. The signal also decreased accompanied by a loss of the enzyme activity under a variety of conditions. When the substrate, catechol, was added to the enzyme under anaerobic conditions, the signal at g = 4.28 disappeared instantaneously. The signal was restored to the original level after all of the substrate was degraded to cis, cis-muconic acid by the addition of air. o-Phenanthroline removed iron from pyrocatechase only when it was added in the presence of sodium dithionite. The decrease in enzyme activity occurred in parallel with the increase in absorbance at 508 mµ due to the formation of a Fe(II)-o-phenanthroline complex. This complex could be separated from enzyme protein by exhaustive dialysis. The colorless inactive protein thus obtained showed essentially no signal in the vicinity of g = 4.2 and contained about 10% of the total iron as determined by the o-phenanthroline method. Upon incubation of this preparation with ferrous ion in the presence of oxygen the signal, as well as the red color, was partially restored with simultaneous reactivation of the enzyme. Other metals including ferric ion did not cause significant reactivation. The removal of iron was prevented by the substrate, catechol, and the concentration of catechol required for half-maximal protection was estimated to be in the same order of magnitude as the Km value for catechol in the catalytic reaction. In light of the above findings, the role of ferric ion in the action of pyrocatechase is discussed.