The effects of insulin on triacylglycerol/fatty acid cycling (fatty acid reesterification) were studied in 12 normal subjects during euglycemic hyperinsulinemia with the use of stable isotope dilution analysis ([2H5]glycerol and [1-13C]palmitate) in combination with indirect calorimetry. During basal conditions, 5.6 +/- 0.6 mumol.kg-1 x min-1 of fatty acid were released of which approximately 3.3 mumol.kg-1 x min-1 were oxidized and approximately 2.2 mumol.kg-1 x min-1 were reesterified. A minority of the recycled fatty acid, (0.8 +/- 0.4 mumol.kg-1 x min-1) never left the intracellular space before being reesterified (intracellular triacylglycerol/fatty acid cycling), whereas the majority (1.2 +/- 0.4 mumol.kg-1 x min-1) were first released into the extracellular space and then reesterified in various organs (extracellular triacylglycerol/fatty acid cycling). In response to insulin, fatty acid release declined by 71% (from 5.6 +/- 0.6 to 1.6 +/- 0.2 mumol.kg-1 x min-1). Fatty acid oxidation (measured by indirect calorimetry) declined by 55% (from 3.3 +/- 0.3 to 1.5 +/- 0.3 mumol.kg-1 x min-1) and total triacylglycerol/fatty acid cycling was completely suppressed (from 2.2 to 0.0 mumol.kg-1 x min-1). Fatty acid release, oxidation, total and extracellular triacylglycerol/fatty acid cycling all correlated positively with plasma fatty acid concentrations. These data showed that insulin profoundly suppressed fatty acid release, oxidation as well as reesterification of those fatty acids that had entered the extracellular compartment. They suggested that physiological concentrations of insulin suppressed extracellular fatty acid reesterification primarily by inhibiting lipolysis.