Ebselen, 2-phenyl-1,2-benzisoselenazol-3(2H)one, and its derivatives were compared for their ability to protect microsomal membranes against iron/ADP/ascorbate-induced lipid peroxidation, measured as low-level chemiluminescence and accumulation of thiobarbituric acid-reactive substances (TBARS). The concentrations of the compounds required to double the lag time of the control with no added antioxidants were 0.13 microM for ebselen, 0.5 microM for the N-pyridyl analog, 0.3-0.7 microM for the selenylsulfides, about 1.0 microM for the selenoxide derivative and 2.0 microM for the sulfur analog of ebselen. The open-chain seleno- and thioether derivatives, on the other hand, exhibited comparatively low abilities to protect the membrane, the lag doubling concentrations for these compounds being 100-1,000 fold higher than that for ebselen. The rate of loss of alpha-tocopherol in the microsomal membrane during peroxidation was significantly diminished in the presence of 0.1-0.5 microM ebselen, while the glutathione adduct of ebselen was equally effective in protecting the loss of alpha-tocopherol. The sulphur analogue and, the benzylated and methylated derivatives of ebselen did not afford protection. Ebselen was without effect in microsomes from vitamin E-deficient rats up to 20 microM, indicative of the dependence of its protective ability upon alpha-tocopherol.