Food matrix and dosage affect isoflavone bioavailability, however, a bread matrix has not been studied. This study evaluated the acute and chronic bioavailability of isoflavones incorporated into breads. In a double-blind, randomized, crossover design, 15 males consumed breads of low-, intermediate-, and high-isoflavone content for 21 days each. Blood from days 1 (fasting and postprandially) and days, 2, 8, 15, and 22 (fasting) and 24-hour urine collections from days -1, 1, 7, 14, and 21 were analyzed for isoflavones. Acute results showed that serum daidzein and genistein increased dose-dependently while Tmax did not change. Chronic results showed that over time, urinary isoflavones did not change while serum isoflavones increased and that treatment effects were inconsistent for urinary and serum isoflavones and depended on isoflavone and study day. These data demonstrate that isoflavone bioavailability in a bread matrix depends on isoflavone dose and varies by specific isoflavone.