In the present study we investigated the relationship between plasma fatty acids (FA) and intramuscular triacylglycerol (IMTAG) kinetics of healthy volunteers. With this aim [U-(13)C]-palmitate was infused for 10 h and FA kinetics determined across the leg. In addition, the rate of FA incorporation into IMTAG in vastus lateralis muscle was determined during two consecutive 4-h periods (2-6 h and 6-10 h). Fifty to sixty per cent of the FA taken up from the circulation were esterified into IMTAG, whereas 32 and 42% were oxidized between 2-6 and 6-10 h, respectively. IMTAG fractional synthesis rate was 3.4 +/- 0.8% h(-1) and did not change between the two 4- h periods, despite an increase in arterial FA concentration (34%, P < 0.01). IMTAG concentration was also unchanged, implying that the IMTAG fractional synthesis rate was balanced by an equal rate of breakdown. FA oxidation increased over time, which could be due to the observed decline in plasma insulin concentration (-74%, P < 0.01). In conclusion, a substantial fraction of the fatty acids entering skeletal muscle in post-absorptive healthy individuals is esterified into IMTAG, due to its high turnover rate (29 h pool(-1)). An increase in FA level, as a consequence of short-term fasting, does not seem to increase IMTAG synthesis rate and pool size.