Postprandial composition of chylomicron triacylglycerols (TAG) and their clearance may be affected by the molecular weight of TAG, their fatty acid (FA) combinations and the positional distribution of FA in TAG. Delayed postprandial TAG clearance is a risk factor for cardiovascular disease. However, due to the complexity of traditional analysis methods, the composition of individual TAG molecules is frequently overlooked. In this study, chylomicron TAG molecular weight distribution and regioisomerism were followed in 10 healthy female volunteers after two fat loads with identical FA composition but different positional distributions (palm oil and transesterified palm oil). An efficient tandem mass spectrometric method of analysis was applied. During the 6-h observation period, the relative concentrations of TAG with 48:2 [48 acyl carbons and 2 double bonds (ACN:DB)], 50:3 and 50:2 decreased, whereas the proportions of 48:0 (tripalmitin), 52:3 and 54:4 remained constant and the proportion of 54:3 (triolein) increased (P < 0.05). The existence of seven regioisomers containing palmitic, oleic and linoleic acids in different sn-positions was studied. The amount of 1,3-dipalmitoyl-2-oleoyl-sn-glycerol was less (P < 0.05) 1.5 h postprandially than at 2-5 h after palm oil, and less (P < 0.05) at 1.5 h than at 2-6 h after transesterified palm oil. This may be an indication of a loss of palmitic acid in the gut. Taken together, TAG molecular weight composition and to a lesser extent, positional distribution, seem to affect the rates of chylomicron TAG clearance in humans.