The relative contents of the fatty acids 14 : 0, 15 : 0 and 17 : 0 in serum and adipose tissue may be used as biological markers of dairy fat intake. However, the determinants of these fatty acids are not fully understood. This study investigates the relationship between these fatty acids and the intake of macronutrients and physical activity in a cross-sectional study of 301 healthy men aged 61-64 years. Dietary intake was assessed using a pre-coded 7 d food record, and physical activity during the previous year was recorded in an interview. Under-reporters of energy intake were identified by the Goldberg cut-off. Fatty acid composition was determined in serum phospholipids (PL) and subcutaneous adipose tissue (AT) from the upper buttock. The relative content of each of 14 : 0, 15 : 0 and 17 : 0 in PL and AT was positively associated with the intake of dairy fat. In addition, all three fatty acids were inversely correlated with alcohol intake, R ranging from -0.28 to -0.53 (P<0.001). The results were not markedly affected when under-reporters (n 88) were excluded from the analyses. In both PL and AT, the relative content of the fatty acids was approximately 5% higher in a group of high physical activity compared with a group of low physical activity, although significant trends were only seen for 14 : 0 in PL and 17 : 0 in AT. The findings suggest that adjustments should be made for alcohol intake when the fatty acids 14 : 0, 15 : 0 and 17 : 0 are applied as markers for dairy fat intake.