To explore the importance of equol on health outcomes in future studies, it was necessary to develop a method to reduce equol production. Female monkeys (n = 22) fed a soy diet were treated twice daily with vehicle (control; n = 4), doxycycline (2.5 mg/kg; n = 4), metronidazole (125 mg/d; n = 3), kanamycin (1000 mg/d; n = 4), vancomycin (100 mg/d; n = 3) or kanamycin+vancomycin (n = 4). Plasma samples were collected 4 h postfeeding at baseline, after 4 wk of treatment and 8 wk after the end of treatment and analyzed for isoflavonoid concentrations. Fecal swabs were collected at baseline and at the end of antibiotic treatment for analysis of Gram(+) and Gram(-) bacterial growth. Equol concentrations were reduced (P < 0.05) compared with baseline by 80, 93, 98 and 99% after treatment with metronidazole (955 +/- 164 vs. 193 +/- 53 nmol/L), kanamycin (545 +/- 211 vs. 37.1 +/- 17.6 nmol/L), vancomycin (607 +/- 163 vs. 8.9 +/- 8.2 nmol/L) and kanamycin+vancomycin (721 +/- 169 vs. 17.4 +/- 17.3 nmol/L), respectively. Daidzein concentrations were increased (P < 0.05) compared with baseline by treatment with doxycycline (336 +/- 87 vs. 576 +/- 76 nmol/L), kanamycin (168 +/- 67 vs. 374 +/- 15 nmol/L), and kanamycin+vancomycin (166 +/- 35 vs. 384 +/- 78 nmol/L). Similar increases (P < 0.05) in dihydrodaidzein were observed after treatment with kanamycin (31.2 +/- 6.2 vs. 479 +/- 188 nmol/L) and metronidazole (56.0 +/- 27.9 vs. 414 +/- 212 nmol/L). Isoflavonoid concentrations returned to baseline values after antibiotic treatment was terminated. Gram(+) bacterial growth was reduced by all treatments, including Control, compared with baseline. In conclusion, treatment with antibiotics resulted in a marked reduction in plasma equol concentrations and altered plasma isoflavonoid patterns in cynomolgus monkeys.