This study tested a hypothesis that soybean lipoxygenase (SLO), a model enzyme, may be capable of generating a glutathione (GSH) conjugate(s) from p-aminophenol (PAP). Horseradish peroxidase was employed as a positive control. GSH depletion or an increase in the absorption at 327 nm with time due to GS-PAP formation was used to quantitate the reaction. The rate of GS-PAP formation was dependent on the incubation time and the amount of SLO and exhibited Km values of 0.44 and 0.71 mM for PAP and H2O2, respectively. Classical inhibitors of lipoxygenase and free radical scavengers markedly decreased the rate of GS-PAP formation in a concentration-dependent manner. PAP-dependent GSH depletion from the reaction medium occurred at a rate of 2.37 +/- 0.18 micromol/min/mg protein. Collectively, the results suggest that lipoxygenase pathway may be involved in the enzymatic formation of GSH conjugate(s) from PAP.