Hog thyroid peroxidase (TPO) was highly purified in order to study the spectral properties and catalytic specificities of its H2O2 compounds in iodothyronine biosynthesis. Purified TPO exhibited a Soret spectrum with an absorption maximum at 410 nm and had an A410/A280 value of 0.55. Protein iodination was only catalyzed under conditions which allowed formation of the transient TPO compound I (Fe(IV)-pi o+). On addition of an equimolar amount of H2O2, TPO formed a stable compound with an absorption maximum at 417 nm. This compound efficiently catalyzed the coupling reaction, but was unable to iodinate proteins. It catalyzed the formation of 1 mol iodothyronines/mol TPO, and therefore retained two oxidizing equivalents per molecule. It is proposed that this compound constitutes a second form of compound I whose structure might be Fe(IV)-Ro, analogous to that of cytochrome c peroxidase compound I. In the presence of an excess of H2O2, it formed TPO-compound III with an absorption maximum at 420 nm. TPO-compound III catalyzed neither the iodination nor the coupling reaction.