During the irreversible inactivation of soybean Fe(III)-lipoxygenase [Fe(III)-LOX] by octadeca-9,12-diynoic acid (ODYA), significant quantities of 11-oxooctadeca-9,12-diynoic acid (11-oxo-ODYA) are formed [Nieuwenhuizen, W. F., et al. (1995) Biochemistry 34, 10538−10545]. To elucidate the inactivation mechanism, a quantitative study into the relationship between the inactivation and 11-oxo-ODYA formation was carried out. The following observations were made. (1) LOX (0.84 μM) was completely inactivated by 10 to 80 μM ODYA. However, at ODYA concentrations greater than 100 μM, LOX was only partially inactivated, and there was no inactivation at all at ODYA concentrations above 750 μM. The average number of turnovers in which 11-oxo-ODYA was formed increased from 1.2 to 12 when the ODYA concentration increased from 1 to 50 μM and then decreased again to 1.2 at 1000 μM ODYA. (2) The enzyme that was not irreversibly inactivated by ODYA was in the Fe(III) form at ODYA concentrations below 10 μM but in the Fe(II) form at ODYA concentrations greater than 100 μM. (3) In the presence of 750 μM ODYA and 25 μM 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid, all of the enzyme was inactivated. On the basis of these results, it is proposed that the dioxygenation product of ODYA is 11-hydroperoxyoctadeca-9,12-diynoic acid (11-HP-ODYA), which can convert Fe(II)-LOX into its Fe(III) form. However, 11-HP-ODYA is converted into 11-oxo-ODYA, which cannot perform the oxidation. It is proposed that the inactivating agent is either 11-HP-ODYA or the 11-peroxy-octadeca-9,12-diynoic acid radical (11-peroxy-ODYA radical), formed from the ODYA radical and O2. The oxidation of Fe(II)-LOX into its Fe(III) form as well as the inactivation of Fe(III)-LOX is competitively inhibited by ODYA.