Lipid peroxidation of rat liver microsomal fractions was monitored by its low-level chemiluminescence in preparations from controls and vitamin-E-deficient animals. Measurements were made (a) of the duration of the lag phase tau0 after initiation with NADPH/iron-ADP and (b) of the slope of the chemiluminescence increase. In microsomes with normal vitamin E (alpha-tocopherol) level the lag phase tau0 was substantially increased by ascorbate; in contrast, even an enhanced peroxidation was observed with ascorbate in vitamin-E-deficient microsomes. Therefore, the ascorbate-mediated protection of microsomalmembranes against lipid peroxidation is dependent on vitamin E in the membrane. In vitamin E deficiency the pro-oxidant effect of ascorbate was abolished when glutathione (GSH) was present. Likewise, GSH does not prolong the lag phase tau0 in vitamin E deficiency. However, GSH (but not cysteine) exerts an antioxidant effect both in controls and in vitamin E deficiency by decreasing the slope of the chemiluminescence increase during lipid peroxidation. The involvement of GSH in an enzyme-dependent mechanism is suggested.