We evaluated the validity of using a single fatty acid tracer to assess total plasma long-chain free fatty acid (FFA) kinetics and the relationship between the rate of appearance (Ra) of fatty acids in plasma and the fatty acid composition of adipose tissue triglyceride (TG). A mixture of [13C]-labeled myristate, palmitate, stearate, oleate, and linoleate was infused in healthy men during basal conditions and during conditions that stimulate (epinephrine infusion) and inhibit (insulin infusion) lipolysis of adipose tissue TGs. Calculated total FFA, Ra based on palmitate, oleate, or linoleate tracers, was within 15% of the measured sum of the individual fatty acid Ra under all conditions, whereas stearate and myristate tracers consistently underestimated and over-estimated total FFA Ra, respectively. The fatty acid Ra profile closely matched the fatty acid profile of subcutaneous adipose tissue TGs during epinephrine infusion, but not during basal conditions and insulin infusion. Our data support the common practice of using labeled palmitate or oleate as fatty acid tracers for assessing total plasma FFA kinetics and suggest that a source of lipids other than adipose tissue TG release fatty acids into the systemic circulation.