Route of nutrient supply is important in regulation of intestinal protein metabolism, because total parenteral nutrition, compared with enteral feeding, leads to profound atrophy. Participation of the fractional rate of protein synthesis (Ks), their degradation in regulation of gut protein balance, and their possible modulation by specific nutrients are the focus of our work. We developed an in situ experimental system that allows controlled exposure of intestinal mucosa to nutrients systemically, luminally, or both. We examined the effects of systemic glucose and amino acid (AA) infusion in overnight-fasted piglets. Jejunal segments within each piglet were simultaneously, luminally perfused with solutions containing various AAs or glucose. Intravenous infusion of glucose increased mucosal Ks by 16% (P < 0.05), whereas intravenous infusion of AA had no effect on Ks. Systemic glucose infusion had no effect on mRNA levels for components of the ubiquitin-proteasome proteolytic pathway. However, levels of these mRNA were reduced by intravenous or luminal AA supply. This effect was greatest (50%) when highest tissue concentrations of AAs were achieved by the simultaneous infusion of AA by both routes (P < 0.05). Our findings suggest that not only is the modulation of protein balance in the intestine in response to nutrients in part attributable to anabolic stimulation of protein synthesis initiated by the systemic appearance of glucose, but a fall in protein degradation is also a likely contributor. AAs appear to be a key factor required to reduce expression of genes connected with proteolysis.