3-(2'-, 3'-, and 4'-Hydroxybenzylidene)-7-methoxychroman-4-one (o-, m-, and p-HBMC) was synthesized for the clarification of the influence of the hydroxyl group at the B ring on the antioxidant activity of homoisoflavonoid. The three homoisoflavonoids used herein can reduce peroxynitrite. p-HBMC exhibited high activity to reduce singlet oxygen. Furthermore, o-, m-, and p-HBMC can scavenge the 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) cationic radical (ABTS(*+)) and 2,2'-diphenyl-1-picrylhydrazyl (DPPH) and galvinoxyl radicals. The rates of o-HBMC trapping of DPPH and galvinoxyl radicals were higher than those of m- and p-HBMC, whereas m-HBMC can trap ABTS(*+) rapidly. o-HBMC was found to possess high activity in the beta-carotene-linoleic acid bleaching test and to protect methyl linoleate against 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH)-induced oxidation efficiently. Finally, o-HBMC served as a prooxidant in Cu(2+)/glutathione (GSH)- and hydroxyl radical-mediated oxidations of DNA. m- and p-HBMC protected DNA against hydroxyl radical-mediated oxidation of DNA effectively, and o- and p-HBMC behaved as antioxidants to protect DNA against AAPH-induced oxidation. Thus, the hydroxyl group attaching to the ortho- and para-positions in the B ring was of importance for the homoisoflavonoid's enhancement of antioxidant activity.