The oxidative activities of primaquine [6-methoxy-8-(4-amino-1-methylbutylamino)quinoline] and its metabolites, the quinone-imine derivatives of 5-hydroxyprimaquine [5-hydroxy-6-methoxy-8-(4-amino-1-methylbutylamino)quinoline] and 5-hydroxydemethylprimaquine [5-hydroxy-6-demethyl-8-(4-amino-1-methylbutylamino)quinoline], 6-methoxy-8-amino quinoline and hydrogen peroxide, were studied on rat erythrocytes in vitro and in vivo. In both cases, the most effective metabolites in oxidizing hemoglobin and depleting non-protein sulfhydryl groups from erythrocytes were the quinone-imine derivatives of the ring-hydroxylated metabolites, 5-hydroxyprimaquine and 5-hydroxydemethyl-primaquine. The latter quinone-imines were shown by light absorption spectroscopy and oxygen consumption studies to be able to oxidize purified rat hemoglobin to methemoglobin but to be unable to react directly with reduced glutathione. In agreement with these results, no radical adduct was detected by electron paramagnetic resonance spectroscopy in incubations of rat erythrocytes with the quinone-imines and the spin-trap 5,5-dimethyl-1-pyrroline-N-oxide; metabolite-derived free radicals were detected instead. Taken together, the results suggest that 5-hydroxyprimaquine and 5-hydroxydemethylprimaquine are important metabolites in the expression of primaquine hemotoxicity, in contrast to 6-methoxy-8-aminoquinoline. Additionally, the results indicate that hydrogen peroxide is the ultimate oxidant formed from the ring-hydroxylated metabolites by redox-cycling of the corresponding quinone-imine derivatives both in vitro and in vivo.