The effect of an organic peroxide, t-butylhydroperoxide (t-BHP), on glutamate uptake was studied in synaptosomes prepared from cerebral cortex. t-BHP inhibited the Na+-dependent glutamate uptake with no change in the Na+-independent uptake. This effect of t-BHP was not altered by addition of Ca2+ channel blockers (verapamil, diltiazem and nifedipine) or PLA2 inhibitors (dibucaine, butacaine and quinacrine). However, the effect was prevented by iron chelators (deferoxamine and phenanthroline) and phenolic antioxidants (N,N'-diphenyl-phenylenediamine, butylated hydroxyanisole, and butylated hydroxytoluene). At low concentrations (＜1.0 mM), t-BHP inhibited glutamate uptake without altering lipid peroxidation. Moreover, a large increase in lipid peroxidation by ascorbate/Fe2+ was not accompanied by an inhibition of glutamate uptake. The impairment of glutamate uptake by t-BHP was not intimately related to the change in Na+-K+-ATPase activity. These results suggest that inhibition of glutamate uptake by t-BHP is not totally mediated by peroxidation of membrane lipid, but is associated with direct interactions of glutamate transport proteins with t-BHP metabolites. The Ca2+ influx through Ca2+ channel or PLA2 activation may not be involved in the t-BHP inhibition of glutamate transport.