To reassess the hypothesis that fatty acid catabolism occurs to completion via β-oxidation, male Sprague–Dawley rats receiving continuous total parenteral nutrition (TPN) including 43% energy as fat were infused with [1-13C]- or [8-13C]triolein. Expired CO2 was collected continuously for 4 h and its 13C: 12C ratio determined by isotope–ratio mass spectrometry. Bicarbonate retention was also assessed over 4 h by infusion of NaH14CO3 and measurement of the expired 14CO2. A possible loss of label from [8-13C]oleic acid from the citric acid cycle via labelled acetyl-CoA without oxidation to CO2 was assessed by infusing further animals with acetate labelled with 14C either at C atoms 1 or 2 and determination of its conversion to expired 14CO2. At isotopic steady state, 63.2 (SE 1.6)% (n 8) of the infused [1-14C]acetate and 46.0 (SE 1.2)% (n 8) of [2-14C]acetate was recovered as expired 14CO2. After correction for bicarbonate retention and non-oxidative isotope loss, 37.3 (SE 1.2)% (n 20) of the [1-13C]triolein was found to have been oxidized, whereas 32.6 (SE 1.0)% (n 20) of the [8-13C]triolein was oxidized (P ≤ 0.01). The lower oxidation of the C atom at position 8 of oleic acid than that at position 1 indicates incomplete oxidative breakdown of the fatty acid after entering β-oxidation.