²H₂O administration has recently been proposed as a simple and convenient method to measure proteins synthesis rates: ²H₂O administration results in deuterium labelling of free amino-acids such as alanine and incorporation into proteins of labelled alanine can then be used to measure protein synthesis rates. We examined first if, during ²H₂O administration plasma free alanine enrichment is a correct estimate of the enrichment in the tissues aminoacids pools used for protein synthesis. We found that after ²H₂O administration, deuterium labelling in plasma free alanine equilibrated rapidly with body water and stable enrichment values are obtained within 20 min. Importantly oral administration of ²H₂O induced no difference of labelling between portal and peripheral circulation, except for the initial 10 minutes after a loading dose. The kinetics of free alanine labelling 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 unlabelled aminoacids from ingested meals do not modify alanine labelling. Calculated synthesis rates of mixed proteins were much higher (20 to 70 fold) in plasma and liver than in muscle and heart. Lastly, comparable replacement rates of ApoB100-VLDL were obtained in humans using the kinetics of incorporation into ApoB100 of infused labelled leucine or of alanine labelled by ²H₂O administration. All these results support ²H₂O as a safe, reliable, useful and convenient tracer for studies of protein synthesis, including proteins with slow turnover rate.