The aim of this work was to determine by ion-exchange liquid chromatography and isotope ratio mass spectrometry the specific 15N enrichment of amino acids in casein and plasma proteins in cows receiving three successive daily oral doses (300, 150 and 150 g) of (15NH4)2SO4 (10 atom per cent isotopic enrichment) and to examine the 15N enrichments obtained with regard to nitrogen transport and metabolism in the lactating cow. To investigate the 15N distribution in amino acids in casein and in plasma proteins, samples of 15N-labelled casein and plasma proteins were extracted either from a pool of several milkings (36–96 h after starting to administer the tracer) or from pooled venous blood (removed on the fourth day after the start of administration) from the four lactating cows. 15N enrichments of the proteins studied, expressed as atoms percent excess, were 0·2509 for casein and 0·0577 for plasma protein. Chromatographic fractionation of the amino acid mixture (protein hydrolysates) resulted in nine groups containing between one and four amino acids: Asp, Ser and Thr; Glu; Pro; Gly, Ala, Val and Met; Ileu and Leu; Tyr; Phe; His and Lys; and Arg. High 15N incorporation was demonstrated in all individual or groups of amino acids studied. In both proteins, Glu appeared to be the most enriched amino acid, Phe and Arg the least enriched. Most aliphatic molecules with a single amino group were highly enriched. The much lower (3·5–7·7-fold) enrichments in plasma protein compared with casein suggest considerable intracellular dilution at the site of liver protein synthesis. Finally, the amino acid separation methods are discussed and suggestions for improving them considered.