We have used stopped-flow rapid reaction methods, employing both fluorescence and absorbance monitoring, together with HPLC analysis of the products to study the activation of soybean 15-lipoxygenase by 13(S)- hydroperoxy-9,11(E,Z)-octadecadienoic acid (13-HPOD). When lipoxygenase is mixed with an equimolar concentration of 13-HPOD, the enzyme undergoes a rapid change in fluorescence. The rate of the change of fluorescence is dependent on the concentration of the 13-HPOD (k = 6.7 x 106 M-1 s-1) and is accompanied by activation of the enzyme. The fluorescence change is not accompanied by any change in the UV absorbance of the 13-HPOD, suggesting no loss of the conjugated diene during enzyme activation, and HPLC analysis of the products of the reaction confirms that the 13-HPOD can be recovered unchanged following this reaction. In the presence of an inhibitor (BWA4C, a hydroxamate inhibitor) that reduces the active-site iron, the 13-HPOD and the inhibitor are destroyed in a peroxidase-like reaction. On the basis of these observations we propose that 13-HPOD binds to the enzyme and facilitates activation of the enzyme, possibly through the formation of a protein radical, and that the 13-HPOD is not changed chemically in this process.