This study examined whether the route of glutamine administration and the simultaneous availability of glucose affect intestinal glutamine metabolism. We measured net substrate exchange rates of glutamine and its nitrogenous products in the isolated vascularly and luminally perfused rat small intestine (a) as a function of glutamine provision from either the vascular or the luminal or simultaneously from both sides and (b) as a function of simultaneous availability of glucose from various routes. When glutamine was provided from the lumen, only 19-32% of absorbed glutamine appeared intact in the venous effluent, but the release of metabolic products was 170 +/- 5 nmol N min-1 g-1. This measure of intestinal glutamine metabolism was unchanged when glutamine was available only in the vascular perfusate (164 +/- 6 nmol N min-1 g-1). It increased, however, to 271 +/- 14 nmol N min-1 g-1 (P < 0.001) when glutamine was available simultaneously from both the luminal and the vascular perfusate. Glutamine consumption (-110 +/- 6 vs. -70 +/- 5 or -91 +/- 5 vs. -73 +/- 7 nmol min-1 g-1; P < 0.05 each) and the production of citrulline (11.4 +/- 0.7 vs. 10.0 +/- 0.8 or 9.8 +/- 0.5 vs. 7.8 +/- 0.4 nmol min-1 g-1; P < 0.05 each) or ammonia (124 +/- 7 vs. 88 +/- 4; P < 0.01 or 78 +/- 4 vs. 68 +/- 5 nmol min-1 g-1) decreased when glucose (vascular or luminal perfusate) became available in addition to glutamine. We conclude that glutamine is utilized by the small intestine very efficiently regardless of the route of administration being enteral or parenteral. The two routes can be used interchangeably to provide the intestinal mucosa with glutamine. Glucose and glutamine may partially substitute each other, most likely for the purpose as a metabolic fuel.