Recent findings that many human chronic diseases are associated with oxidative stresses have instigated the search for dietary antioxidants. Many phytochemicals, particularly phenolic compounds, have been found to possess strong antioxidant activity and reduce the risks of those diseases. Isoflavones, a special phenolic group found in soybean, have been found to act as antioxidants in some model systems. This study investigated the isoflavone content in a unique nonedible tree fruit, Osage orange [Maclura pomifera (Raf.) Schneid], and methods for the extraction, identification, and quantification of the two major isoflavones, osajin and pomiferin, were developed. The ethyl acetate extract contained 25.7% osajin and 36.2% pomiferin, and the two isoflavones were at 9.5 g kg(-1) of fresh Osage orange. Two model systems, FRAP and beta-CLAMS, were used to measure the antioxidant activity of these two isoflavones. Pomiferin was found to be a strong antioxidant in both systems, comparable to the antioxidant vitamins C and E and the synthetic antioxidant BHT. Osajin and the two soybean isoflavones (genistein and daidzein) showed no antioxidant activity. Although the Osage orange fruit is not a food source, it is considered to be safe and, therefore, a potentially good source of an antioxidant nutraceutical and functional food ingredient.