The unfolding of soybean lipoxygenase-1 during urea denaturation has been followed by activity assays and fluorescence measurement. The presence of stable intermediates during unfolding for both ferrous and ferric forms of lipoxygenase-1 were observed. In the presence of 6.0 M urea, the unfolding of soybean lipoxygenase-1, as monitored by fluorescence intensity, is a triphasic process, while the inactivation of the enzyme shows a single-phase kinetics. The rate constant of inactivation is consistent with that of the fast conformational change of the enzyme. Based on these, a minimal scheme containing two intermediates was proposed tointerpretthe unfolding of lipoxygenase-1 induced by urea.