13(S)-Hydroperoxyoctadeca-9(Z),11(E),15(Z)-trienoic acid (13-HPOT) was used to probe the mechanism of the hydroperoxide O-O bond cleavage catalyzed by solubilized and partially purified soybean peroxygenase. When reacted with this ferrihemoprotein, it was converted to 13(S)-hydroxyoctadeca-9(Z),11(E), 15(Z)-trienoic acid (13-HOT) and a single epoxide regio-isomer, i.e. 15,16-cis-epoxy-13(S)-hydroxyoctadeca-9(Z),11(E)-dienoic acid (15,16-EHOD). In the absence of co-oxidizable substrates, such as oleic acid or thiobenzamide, this latter compound accounted for about two-thirds of the reaction products. 13-HOT and 15,16-EHOD are products of heterolytic scission of the O-O bond of 13-HPOT; no products arising by homolytic scission could be detected. Therefore, soybean peroxygenase catalyzes hydroperoxide reduction exclusively by a heterolytic mechanism leading to a ferryl-oxo complex analogous to peroxidase compound I. In similar experiments, 13(S)-hydroperoxyoctadeca-9(Z),11(E)-dienoic acid gave 13(S)-hydroxyoctadeca-9(Z),11(E)-dienoic acid and 9,10 epoxy-13(S)-hydroxyoctadec-11(E)-enoic acid. Experiments with 18O-labeled 13-HPOT indicated that about 83% of the oxygen atom incorporated into the epoxide group of 15,16-EHOD, originated from the hydroperoxide group. Moreover, using mixtures of unlabeled and 18O-labeled 13-HPOT, it was established that this transfer takes place predominantly (about 3:1) by an intramolecular process. In the intermolecular reaction 13-HOT, formed after reduction of the hydroperoxide, diffuses from the active site and, after reassociation, is epoxidized at the 15,16-double bond. A unifying mechanistic scheme, which takes into account all of the reactions catalyzed by the peroxygenase, is proposed.