BACKGROUND/OBJECTIVES: Isothiocyanate (ITC) is formed via the hydrolysis of glucosinolates by myrosinase, found in cruciferous vegetables. Although myrosinase is inactivated by the cooking process, no studies have incorporated the effect of cooking into the estimation of dietary ITC intake or evaluated the validity. We evaluated the validity of dietary ITC intake estimated from a food frequency questionnaire (FFQ), and urinary ITC levels using 24 h urine samples or a WFR (weighed food record), and evaluated the reproducibility of dietary ITC in two FFQs administered at an interval of 1-year. SUBJECTS/METHODS: The JPHC-NEXT Protocol Area included a total of 255 middle-aged participants across Japan. We calculated dietary ITC intake from WFR and two FFQs by assuming that cooked cruciferous vegetables contain zero ITC. Urinary ITC excretion was measured at two points during summer and winter. The validity and reproducibility of dietary ITC intake estimated by FFQ were assessed using Spearman's correlation coefficients. RESULTS: Although we observed a moderate correlation between dietary ITC intake derived from a 12-day WFR and urinary ITC excretion, notwithstanding the cooking process, the correlation between dietary ITC intake estimated by FFQ and mean urinary ITC excretion was low. However, the correlation was improved when we compared urinary ITC excretion and a 3-day WFR or FFQ collected during winter. Our FFQ showed good reproducibility. CONCLUSION: Although seasonality is a critical factor, dietary ITC intake estimated using an FFQ showed moderate validity and reproducibility and can be used in future epidemiological studies.