We studied endogenous substrates for P-glycoprotein (P-gp) in an oxidative reaction mixture of ceramides, phospholipids, sphingolipids, or GM1-gangliosides (GM1-G). Extracts from the reaction mixture of galactocerebrosides (GalCer), sphingomyelin (SM) , lactocerebrosides (LactoCer), and asolectine (AS) with 0.3% hydrogen peroxide exhibited significant ATPase activity of P-gp of 7.6, 7.8, 5.3, and 4.7 nmol/min/mg protein, respectively, at a concentration of 10 μg equivalent/ml, but not GalCer, SM, LactoCer, and AS themselves. Meanwhile, both GM1-G and its oxidized product showed ATPase activity of 3.7 nmol/min/mg protein at a concentration of 0.75 μM. Phosphatidylcholine, phosphatidylethanolamine, phophatidylserine, triglyceride, and cholesterol did not show P-gp activity. When reactive oxygen species, such as hydrogen peroxide, exceed the ability of antioxidant defense systems to remove it from living cells, SM, GalCer, LactoCer, and AS could react with it; therefore, it is possible for these oxidized lipids to play as substrates for Pgp in living cells. This finding should be a milestone to search a new physiological P-gp function.