Proteolytic and cataplerotic sources of hepatic glutamine were determined by (2)H NMR analysis of urinary phenylacetylglutamine (PAGN) (2)H-enrichments in eight healthy subjects after (2)H(2)O and phenylbutyric acid ingestion. Body water enrichment was 0.49+/-0.03%. PAGN was enriched to lower levels with significant differences between the various glutamine positions. PAGN position 2 enrichment=0.33+/-0.02%; 3R=0.27+/-0.02%; 3S=0.27+/-0.02% and position 4=0.17+/-0.01%. Position 3R,S enrichments are conditional with the net conversion of citrate to glutamate and are therefore markers of cataplerosis. From the ratio of positions 3R,S to body water enrichment, 55+/-3% of hepatic glutamine was derived from cataplerosis and 45+/-3% from proteolysis. In conclusion, enrichment of PAGN 3R,S hydrogens relative to that of body water reflects the contribution of cataplerotic and proteolytic sources to hepatic glutamine.