Whole-body and gastrointestinal tract (GIT) metabolism of [5-15N]glutamine were monitored in lambs (33 kg live weight) fasted for 24 h. Animals were previously prepared with vascular catheters across the mesenteric-(MDV) and portal-drained viscera (PDV) to permit quantification of mass and isotopic transfers of metabolites by arterio-venous difference. Continuous infusions of [5-15N]glutamine into the jugular vein were conducted for 10 h and integrated blood samples withdrawn over 75 min intervals for the last 5 h of infusion. The lambs were then killed and portions from various tissues of the digestive tract and other body organs removed for determination of 15N enrichment in RNA, DNA and protein (the latter obtained by difference using total acid-precipitable N). Whole-body glutamine flux was 108 μmol/min of which 23 and 47 % could be attributed to MDV and PDV metabolism (P < 0·001) respectively. There was a small net production of glutamine across the MDV. GIT blood-flows and NH3 production were partitioned 3:2 between MDV and non-MDV components. Less than 5 % of the NH3 produced was derived from the amido-N of glutamine, while across the small intestine (MDV) 26 % of the glutamine flux was converted to NH3, compared with 18 % for non-MDV transfers. The 15N enrichments in protein were of the order jejunum > duodenum > ileum with mucosal cells more labelled than serosal (P < 0·001). Lesser enrichments were observed for other GIT tissues (abomasum > caecum > rumen) while liver and lymph were comparable with the abomasum; kidney, spleen and muscle were lower still (P < 0·05). Enrichments of RNA were similar to that of protein and followed the same pattern, except for higher relative values for liver, spleen and lymphoid tissue. The lowest enrichments were observed for DNA, but again the pattern order was similar except for increased label in lymph, caecum and the spleen. For the MDV there was reasonable agreement between 15N-disappearance as glutamine and appearance in NH3 (24 %), protein (81 %), RNA (3·6 %) and DNA (2·1 %). For the total PDV there was a shortfall (−12 %), however, which may be due to losses in lumen components. These results show the importance of the GIT as a contributor to total glutamine plasma flux, but indicate a lesser reliance on glutamine metabolism by the digestive tract of the ruminant compared with observations from non-ruminants.