Quantitatively accurate measurements of plasma free fatty acid (FFA) turnover can be made if the FFA tracer is infused intravenously and arterial blood is sampled. To be certain that arterialized-venous blood provides comparable information, plasma FFA concentration and specific activity were measured in both arterial and arterialized-venous blood in 19 volunteers undergoing infusion of [1-14C]palmitate infusion before and during a euglycemic, hyperinsulinemic clamp. Heated hand vein blood was obtained using either the heated box (n = 9 subjects) or heating pad (n = 10 subjects) technique. The heated box arterialized-venous blood had palmitate concentration and specific activity equal to arterial blood. The use of the heating pad resulted in small (3%), but statistically significant (P less than .05), underestimates of palmitate specific activity. In addition, plasma lactate concentrations were significantly higher in heated hand vein blood than arterial blood. In summary, heated hand vein blood is a satisfactory alternative to arterial blood for tracer studies of FFA metabolism.