To validate a self-administered food frequency questionnaire (FFQ) for the estimation of dietary intake of folate, vitamins B6 and B12, we compared measures of these vitamins by the FFQ and plasma concentrations among 87 male volunteers aged 40-89 years in the Japan Public Health Center-based prospective study (JPHC Study). No men used folate, vitamins B6 or B12 as supplements. There was a moderate association between energy-adjusted dietary intake and plasma concentration for folate and vitamin B6. The Spearman correlation coefficient was 0.26 and 0.23, respectively, in the total samples. These correlation coefficients were slightly higher when men in the Ishikawa PHC area were excluded from the analysis; the correlation coefficient was 0.29 for folate and 0.26 for vitamin B6. No significant correlation was found between dietary vitamin B12 intake and serum B12 concentration; the correlation coefficient was 0.06 in the total sample and 0.15 when the Ishikawa men were excluded from the analysis. In conclusion, we found that the FFQ used for the 5-year follow-up survey of JPHC Study could reasonably rank individuals by dietary intake of folate and vitamin B6, but not vitamin B12, among Japanese community-dwelling population samples.