Instability and limited solubility hampers the inclusion of free glutamine in aqueous preparations intended for clinical nutrition. This drawback can be overcome by using synthetic stable and highly soluble glutaminecontaining dipeptides. Rapid utilization in the target tissue is a prerequisite for their rational use. Hydrolytic capacity of rat small intestine against L-alanyl-L-glutamine and glycyl-L-glutamine was assessed by incubating mucosa cell fractions (brush border and basolateral membrane vesicles, soluble fraction) under controlled and standardized conditions. Subsequently dipeptide disappearance and the simultaneous liberation of the constituent free amino acids were monitored (liquid chromatography) enabling estimation of the Michaelis constants and the maximum velocity of the reaction.The estimated Michaelis constants (Eadie-Hofstee plot) for membrane-bound peptidases appeared to be lower for alanyl-glutamine than those for glycyl-glutamine (brush border membrane: 0.99 + 0.23 vs. 4.59 + 2.01 mmol/L; basolateral membrane: 1.02 f 0.38 vs. 4.80 f 0.89 mmoWL). The estimated capacity for hydrolysis, expressed per mg mixed protein, was 3-to j-fold higher for alanyl-glutamine than for glycyl-glutamine in all preparations.The results indicate that mammalian intestinal mucosa is well equipped to enable an eficient hydrolysis of glutamine-containing dipeptides either delivered luminally or intravenously.