We investigated the validity and reproducibility of the FFQ used in the Dutch European Investigation of Cancer and Nutrition cohort, in order to rank subjects according to intakes of fatty acid classes and individual fatty acids. In total, 121 men and women (23–72 years) filled out three FFQ at 6-month intervals between 1991 and 1992. As a reference method, they filled out twelve monthly 24-h dietary recalls (24HDR) during the same year. Intra-class correlation coefficients for the FFQ showed moderate to good reproducibility across all fatty acids (classes and individual) in men (0·56–0·81) and women (0·57–0·83). In men, Spearman’s correlation coefficients (r
s) for the FFQ compared with the 24HDR indicated moderate to good relative validity (r
s=0·45–0·71) for all fatty acids, except for arachidonic acid and marine PUFA (r
s<0·40). In women, relative validity was moderate to good for MUFA and trans-fatty acids (TFA) and the majority of SFA (r
s=0·40–0·66), was fair for the short-chain SFA and lauric acid (r
s=0·30–0·33) and was fair to moderate for PUFA (r
s=0·22–0·47). Bland–Altman plots showed good agreement between the FFQ and 24HDR, and proportional bias for fatty acids with very low intakes. In conclusion, the FFQ showed good reproducibility for subject ranking based on intakes of fatty acids (classes and individual). The relative validity measures indicated that the FFQ is an adequate tool to rank subjects according to intakes of high-abundant fatty acids, but less for low-abundant fatty acids.