Chronic high protein consumption leads to increased concentrations of NH₄ ⁺/NH₃ in the colon lumen. We asked whether this increase has consequences on colonic epithelial cell metabolism. Rats were fed isocaloric diets containing 20% (P20) or 58% (P58) casein as the protein source for 7 days. NH₄ ⁺/NH₃ concentration in the colonic lumen and in the colonic vein blood as well as ammonia metabolism by isolated surface colonic epithelial cells were determined. After 2 days of consumption of the P58 diet, marked increases of luminal and colonic vein blood NH₄ ⁺/NH₃ concentrations were recorded when compared with the values obtained in the P20 group. Colonocytes recovered from the P58 group were characterized at that time and thereafter by an increased capacity for L-ornithine and urea production through arginase (p<0.05). L-ornithine was mostly used in the presence of NH₄Cl for the synthesis of the metabolic end product, L-citrulline. After 7 days of the P58 diet consumption, however the ammonia metabolism into L-citrulline was found lower (p<0.01) when compared with the values measured in the colonocytes recovered from the P20 group despite any decrease in the related enzymatic activities (i.e. carbamoyl-phosphate synthetase I and ornithine carbamoyl transferase). This decrease was found to coincide with a return of blood NH₄ ⁺/NH₃ concentration in colonic portal blood to values close to the one recorded in the P20 group. In response to increased NH₄ ⁺/NH₃ concentration in the colon, the increased capacity of the colonocytes to synthesize L-ornithine is likely to correspond to an elevated L-ornithine requirement for the elimination of excessive blood ammonia in the liver urea cycle. Moreover, in the presence of NH₄Cl, colonocytes diminished their synthesis capacity of L-citrulline from L-ornithine, allowing a lower cellular utilization of this latter amino acid. These results are discussed in relationship with an adaptative process that would be related to both inter-organ metabolism and to the role of the colonic epithelium as a first line of defense towards luminal NH₄ ⁺/NH₃ concentrations.