The validation of dietary total fat measurements has been elusive because no specific biomarker exists. In metabolic studies with controlled diets, plasma fasting triglyceride levels are reduced with higher fat intake and can thus serve as an "alloyed gold" standard. Participants in this cross-sectional analysis were 269 men aged 47-83 years from the Health Professionals Follow-up Study who completed a semiquantitative food frequency questionnaire and provided fasting blood specimens in 1994. In a multiple regression analysis adjusted for age; smoking; alcohol consumption; physical activity; body mass index; and intakes of protein, dietary fiber, and total energy, total fat intake was inversely associated with fasting triglycerides (for a fat increase of 1% of energy, triglyceride levels were lower by 2.5% (95% confidence interval: -3.7 to -1.3%, p = 0.0002)). For reported fat intakes of 20% or less of energy, the geometric mean fasting triglyceride level was 179, and for more than 40% of energy, it was 102 mg/dl. In addition, as predicted by metabolic studies, the inverse association between dietary fat and fasting triglyceride level was much stronger among overweight men than among men with a BMI of less than 25. These data provide additional evidence that informative measurements of dietary fat can be obtained by carefully constructed food frequency questionnaires.