H₂O administration has recently been proposed as a simple and convenient method to measure protein synthesis rates. ²H₂O administration results in deuterium labeling of free amino acids such as alanine, and incorporation into proteins of labeled alanine can then be used to measure protein synthesis rates. We examined first whether during ²H₂O administration plasma free alanine enrichment is a correct estimate of the enrichment in the tissue amino acid pools used for protein synthesis. We found that, after ²H₂O administration, deuterium labeling in plasma free alanine equilibrated rapidly with body water, and stable enrichment values were obtained within 20 min. Importantly, oral administration of ²H₂O induced no difference of labeling between portal and peripheral circulation except for the initial 10 min after a loading dose. The kinetics of free alanine labeling were comparable in various tissues (liver, skeletal muscle, heart) and in plasma with identical plateau values. We show next that increased glycolytic rate or absorption of unlabeled amino acids from ingested meals do not modify alanine labeling. Calculated synthesis rates of mixed proteins were much higher (20- to 70-fold) in plasma and liver than in muscle and heart. Last, comparable replacement rates of apoB100-VLDL were obtained in humans by using the kinetics of incorporation into apoB100 of infused labeled leucine or of alanine labeled by ²H₂O administration. All of these results support ²H₂O as a safe, reliable, useful, and convenient tracer for studies of protein synthesis, including proteins with slow turnover rate.