Immune cell functions could be evaluated in vivo by measuring their specific protein fractional synthesis rates (FSR). Using stable isotope dilution techniques, we describe a new method allowing the simultaneous in vivo assessment of FSR in two leukocytes populations, in healthy humans subjects using small blood samples. Peripheral blood mononuclear cells (PBMC) and polymorphonuclear neutrophils (PMN) FSR were measured during a prime continuous IV infusion of L[1-¹³C]leucine. Immune cells from 6 ml whole blood were isolated by density gradient centrifugation. In a first study, we calculated the FSR by using plasma [¹³C]-leucine or [¹³C]-alpha-ketoisocaproate (KIC) enrichments as precursor pools. In a second study, we compared protein FSR in leukocytes by using at immediate precursor pool either the enrichment of intracellular or plasma free [¹³C]-leucine. The present approach showed a steady state enrichment of plasma and circulating immune cell free [¹³C]-leucine precursor pools. The linearity of labelled amino acid incorporation rate within mixed PBMC and PMN proteins was also verified. Postabsorptive protein FSR in PBMC was 4.09±0.39%/d and 1.44±0.08%/d in PMN by using the plasma [13C]-KIC as the precursor pool. The difference between PBMC and PMN FSR was statistically significant whatever the precursor pool used, suggesting large differences in their synthetic activities and functions. The use of plasma [¹³C]-KIC pool led to overestimate leukocyte FSR in comparison with the intracellular precursor compartment (PBMC: 6.04±0.94%/d and PMN: 2.98±0.30%/d). Hence, the intracellular free amino acid pool must be used as precursor to obtain reliable results. In conclusion, it is possible to assess immune cell metabolism in vivo in humans using small blood samples to have a direct indication of their metabolic activity in various clinical situations and in response to regulating factors.