Iron K-edge X-ray spectroscopy (XANES and EXAFS) was used to study iron coordination in frozen solutions of soybean lipoxygenase-1 (SLO). The intensity of the 1s-->3d pre-edge transition of native iron(II) lipoxygenase is greater than what was found for six-coordinate high-spin iron(II) model complexes, but comparable to that of a five-coordinate model. This and a relatively short average bond length determined by EXAFS (2.13 A) indicate that the native lipoxygenase in our frozen samples is five-coordinate, excluding possible bonds longer than 2.5 A. The coordination of the iron(II) in native lipoxygenase changes when methanol (as low as 0.1%) or glycerol (20%) is added to the buffer prior to freezing. The addition of methanol diminishes the pre-edge transition and increases EXAFS-derived bond lengths by 0.04 A, indicating a change to six-coordination. The small pre-edge feature in active iron(III) lipoxygenase suggests six-coordination. EXAFS indicates a short, 1.88 A Fe-O bond, which, given other spectroscopic and crystallographic evidence, is assigned to coordinated hydroxide. The average of the remaining bond lengths is 2.11 A. The iron coordination in iron(III) lipoxygenase is less affected by the presence of alcohols than is the site in the iron(II) enzyme. Bond valence sums indicate that the bond lengths for lipoxygenase derived from our EXAFS analyses are comparable to those of crystallographically characterized model complexes. The flexibility of the coordination number in SLON (native SLO) and the presence of an [FeIIIOH]2+ unit in SLOA (active SLO) are of possible mechanistic importance.