The isoflavone compositions and concentrations in the leaf, flower, petiole, and stem of 13 red clover cultivars were studied using high-performance liquid chromatography coupled with a diode array and a mass spectrometric detector with negative electrospray ionization. Different cultivars showed significantly different concentrations of individual and total isoflavones. The leaf contained the highest overall concentration, followed by the stem, petiole, and flower. Biochanin A and formononetin were the predominant isoflavones in all cultivars and all parts, along with eight other minor aglycones, daidzein, genistein, glycitein, irilone, orobol, pratensein, pseudobaptigenin, and prunetin, and four minor malonylglycosides, genistein-7-glucoside-6' '-malonate, orobol-7-glucoside-6' '-malonate, formononetin-7-glucoside-6' '-malonate, and biochanin A-7-glucoside-6' '-malonate. The isoflavone compositions and concentrations were also found to be different between red clover parts harvested at the early bud stage and the late flowering stage. Sample storage and handling prior to analysis were also found to be important. Samples in this study were kept at -5 degrees C for a few days before being freeze-dried and were found to contain mainly the aglycones of isoflavones. This may actually be an advantage in that "natural" and more bioactive isoflavones can be obtained without using chemical hydrolysis. Findings in this study therefore provide important information for developing isoflavone-rich red clovers and for optimizing harvest timing and choosing the right part of the red clover plant.