To determine whether circulating citrulline can be manipulated in humans, and, if so, whether citrulline availability affects the levels of related amino acids, nitric oxide, urinary citrulline, and urea nitrogen, 10 healthy volunteers were studied on 3 separate days: 1) under baseline conditions; 2) after a 24-h treatment with phenylbutyrate, a glutamine "trapping" agent; and 3) during oral L-citrulline supplementation (0.18 g.kg-1.d-1). Plasma, erythrocyte, and urinary citrulline concentrations were determined by GCMS at 3-h intervals between 1100h and 2000h on each study day. Regardless of treatment, erythrocyte (RBC) citrulline was lower than plasma citrulline (RBC/plasma ratio of 0.60±0.04), and urinary citrulline excretion accounted for <1% of the citrulline load filtered by kidney. Phenylbutyrate induced an 7% drop in plasma glutamine (p=0.013), and 18±14% (p<0.0001) and 19±17% (p<0.01) declines in plasma and urine citrulline, respectively. Oral L-citrulline administration was associated with: 1) a rise in plasma, urine and RBC citrulline, (39±4 vs. 225±44 µmol/L, 0.9±0.3 vs. 6.2±3.8 µmol/mmol creatinine, and 23±1 vs. 52±9 µmol/L, respectively); and 2) a doubling in plasma arginine level, without any change in blood urea or urinary urea nitrogen excretion, and thus enhanced nitrogen balance. We conclude that: 1) depletion of glutamine, the main precursor of citrulline, depletes plasma citrulline; 2) oral citrulline can be used to enhance systemic citrulline and arginine availability, as citrulline is bioavailable, and very little citrulline is lost in urine; and 3) further studies are warranted to determine the mechanisms by which citrulline may enhance nitrogen balance in vivo in humans.