Despite considerable interest in the physiologic effects of isoflavones, the in vivo bioavailability of the most common isoflavone forms, malonylglucoside conjugates, has not been determined. Differences in the bioavailability of malonylglucosides compared with the nonconjugated β-glucoside forms may explain the inconsistent findings regarding the physiologic effects of isoflavones. Therefore, our objective was to determine the effect of malonyl- conjugation on isoflavone bioavailability in an animal model. Malonylgenistin and malonyldaidzin, and their corresponding nonconjugated glucosides, were extracted from soy grits and purified using liquid chromatography. Purity of the isolated forms was confirmed by nuclear magnetic resonance analysis. Male rats were gavaged with malonylgenistin, genistin, malonyldaidzin, or daidzin at a dose of 100 μmol/kg body weight. Blood and urine samples were collected at time intervals ranging from 0 to 48 h. Isoflavone metabolites in plasma and urine were determined using stable isotope dilution- liquid chromatography/mass spectrometry. Comparisons of pharmacokinetic variables were made between nonconjugated and conjugated glucosides and over time of plasma collection. The areas under the time-concentration curve of the metabolites in the plasma obtained after the administration of nonconjugated β-glucosides were 1 to 6 times higher than those of their respective malonylglucosides (P ≤ 0.05). Additionally, maximum plasma concentration and urinary excretion of isoflavone metabolites were significantly higher (1-9 times; P ≤ 0.05) after the administration of nonconjugated β-glucosides. To our knowledge, these results demonstrated, for the first time, that nonconjugated β-glucosides are relatively more bioavailable than their respective malonylglucosides. These differences in the bioavailability of conjugated and nonconjugated β-glucosides should be considered in future studies focused on the bioactivity of isoflavones.