Objective: The aim of this study was to examine the distribution of trans fatty acids (TFA) in plasma lipid classes and the relationship with dietary intake of TFA. Design: After a 2 week baseline (habitual) diet, all subjects consumed a moderate fat (MF) diet for 3 weeks with the fat being derived mainly from margarine and the rest from lean beef, and then a very low fat (VLF) diet for 3 weeks with the TFA being derived only from the lean beef. Blood samples were collected 2 days prior to the end and also on the last day of each dietary period. Setting: Deakin Institute of Human Nutrition, Deakin University, Geelong, Australia. Subjects: Ten free-living mildly hypercholesterolaemic subjects aged 22-66 were recruited in Geelong. Outcome measures: TFA intake was calculated from analyses of Australian margarines, butter, lean meat and animal fat. The TFA in plasma lipid fractions were separated by AgNO3 thin-layer chromatography and quantitated by capillary gas-liquid chromatography using internal standards. Results: The phospholipid (PL) fraction contained more than 60% of the trans-18:1 isomers in the plasma lipids in all subjects. On the baseline diet, the predominant positional isomer of trans-18:1 in PL was Δ11, whereas in the other lipid classes it was the Δ9 isomer. The concentration of the Δ9 isomer increased on the MF diet, particularly in the PL fraction, while the concentration of the Δ11 isomer decreased in all fractions. On the VLF diet, the total TFA level decreased by approximately 50%, mainly due to decreases in the TFA isomers in the PL and TG fractions. Changes in plasma total and PL TFA, PL Δ9, Δ10 and Δ11 were strongly correlated with dietary TFA intake (P < 0.0001). There were also significant association between dietary TFA intake and PL Δ12 (P = 0.003), triacylglycerol Δ9 (P = 0.009), Δ11 (P = 0.0005), total triacyglycerol (P = 0.023) and free fatty acid TFA (P = 0.042). Conclusions: The results suggest that the measurement of trans-18:1 in plasma PL and TAG, and plasma total TFA could be used to estimate the intake of TFA.