From the n-hexane fraction of the fruits of Vitex agnus-castus, two labdane-type diterpenes, vitetrifolin B and C, were isolated by means of multiple chromatographic separations, together with the previously identified rotundifuran, vitexilactone and the sesquiterpene spathulenol. From the EtOAc fraction, eupatorin was identified for the first time, besides the known casticin, penduletin, vitexin and orientin. The n-hexane, EtOAc and MeOH–H2O fractions of the MeOH extract of Agni-casti fructus were subjected to in vitro antioxidant assays. The EtOAc extract displayed a significant concentration-dependent effect when tested by 1,1-diphenyl-2-picrylhydrasyl (DPPH) free radical assay (IC50 = 68 µg/mL) and against the autooxidation of a standard rat brain homogenate (IC50 = 14 µg/mL). The MeOH–H2O fraction was less active with 3643 µg/mL (DPPH test) and IC50 = 125 µg/mL (rat brain homogenate), while the n-hexane phase proved to be inactive. The main flavonoid constituents of the EtOAc extract, casticin, vitexin and orientin were assayed for antioxidant activity and found that only casticin possesses a marked lipid peroxidation inhibitory effect (IC50 = 0.049 mm) compared with that of the positive control ascorbic acid (IC50 = 0.703 mm). Copyright © 2007 John Wiley & Sons, Ltd.