OBJECTIVE: To evaluate the reliability and validity of a food-frequency questionnaire (FFQ) and database designed to quantify phytoestrogen consumption. METHODS: This study included 195 members of the California Teachers Study (CTS) cohort who, over a 10-month period, completed four 24-h dietary recalls, a pre- and post-study FFQ, and provided two 24-h urine specimens. Participants (n = 106) in a parallel study (and 18 women who dropped out of the long-term study) completed a single recall and FFQ, and provided one 24-h urine specimen. Urinary phytoestrogens were determined using liquid chromatography-mass spectrometry. Reliability and validity were evaluated using Shrout-Fleiss intraclass correlations and energy-adjusted deattenuated Pearson correlations, respectively. RESULTS: Correlations reflecting the reproducibility of the FFQ phytoestrogen assessment ranged from 0.67 to 0.81. Validity correlations (FFQ compared to dietary recalls) ranged from 0.67 to 0.79 for the major phytoestrogenic compounds (i.e., daidzein, genistein, and secoisolariciresinol) and 0.43 to 0.54 for the less common compounds. Compared to urinary levels, validity correlations ranged from 0.41 to 0.55 for the isoflavones and 0.16 to 0.21 for total lignans. CONCLUSION: Our isoflavone assessment is reproducible, valid, and an excellent tool for evaluating the relationship with disease risk in non-Asian populations. Further research is needed before these tools can accurately be used to assess lignan consumption.