The magnetic susceptibilities of yellow soybean lipoxygenase-1 before and after anaerobic reduction with the substrate linoleic acid have been studied over the temperature range 10–170 K. Theoretical temperature dependencies of the magnetic susceptibility, calculated by means of a spin Hamiltonian, have been fitted to the experimental data. The conversion of native into yellow enzyme (involving iron oxidation) by (13 S-hydroxy-9 Z,11 E-octadecadienoic acid ((13 S-HPOD) was found by EPR and light-absorption studies to be progressively less effective at increasing enzyme concentrations. A theoretical model could be fitted to the experimental susceptibility data of a yellow sample by assuming the EPR-invisible iron fraction to contain high-spin Fe(II) with its zero-field splitting parameter D in either of the range 8 ± 3 cm −1 or −6.5 ± 1.5 cm −1. Our results indicate a ligand environment of axial symmetry, probably with a rhombic distorsion, and suggest that the EPR-invisible iron fraction has remained Fe(II), just like in the native enzyme. There is no evidence that the EPR-invisible iron is due to high-spin Fe(III) antiferromagnetically coupled to a radical. Our results for the anaerobically substrate-treated yellow lipoxygenase are consistent with the presence of high-spin Fe(II) with D in either of the ranges 12.5 −2 +1.5 cm −1 or −12 −2 +3 cm −1. It is concluded that linoleic acid can reduce the iron of yellow lipoxygenase. The present results are definite evidence for a valence change during the anaerobic reaction. Reduction of iron by linoleic acid is possibly also a feature of the dioxygenation reaction.