As arginine plays a key role in the regulation of liver ureagenesis, we hypothesised that a modulation of enzymes involved in arginine metabolism within the intestine contributes to the regulation of N homeostasis according to protein supply. Our aim was to study the influence of variations in protein or amino acid (AA) supply on intestinal arginase, glutaminase, ornithine aminotransferase (OAT), argininosuccinate lyase and argininosuccinate synthetase. We evaluated in vivo in rats the responses of these enzymes to short-term (ST, 16 h) and long-term (LT, 15 d) variations in dietary protein (10, 17 or 25 % protein diet). In addition, in order to test whether these responses could involve a direct action of AA on the gene expression and activity of these enzymes, Caco-2/TC7 cells were cultured for 3 d with increasing AA concentrations. In vivo, in the ST, both high- and low-protein diets increased arginase activity in the intestinal mucosa (ST25 %: 46 (sem 2) μmol/g per min and ST10 %: 46 (sem 2) μmol/g per min v. ST17 %: 36 (sem 3) μmol/g per min, P < 0·05). In the LT, OAT expression was increased in the LT10 % group (+277 %, P < 0·05) compared with the LT17 % group. Caco-2/TC7 cells showed inverse relationships between AA supply and arginase (P = 0·058) and OAT (P = 0·035) expressions. The present study demonstrates the regulation of intestinal arginase and OAT expressions in response to protein supply. Our in vitro experiments further indicate a direct AA-induced regulation of the mRNA abundance of these enzymes. In situations of limited protein supply, this regulation would increase intestinal arginine catabolism and, possibly via a decrease in arginine portal release, decrease hepatic AA oxidation, thus promoting N sparing.