The capacities of viable colonic epithelial cells to metabolize glucose, glutamine and n-butyrate were studied in 30-kg pigs adapted to a high fiber (12% sugar beet fiber) or a low fiber diet. Glucose and glutamine were extensively utilized but predominantly not oxidized, whereas n-butyrate oxidation accounted for 45% of n-butyrate metabolism and was not greatly affected by the presence of glucose or glutamine. With both diets, glycolysis was the major pathway accounting for glucose disappearance. There was a sparing effect of n-butyrate on both glycolysis and glucose oxidation. Moreover, the glycolytic capacity was 25% lower in pigs fed the high fiber diet. Data suggest that 6-phosphofructo-1-kinase could be the regulatory step in glycolysis. Nevertheless, its maximum activity was not affected by the diet or by the presence of n-butyrate. Glutamine metabolism was slightly affected by fiber in the diet and by the presence of n-butyrate. In addition to CO2, butyrate was converted into ketone bodies. Glucose and glutamine did not substantially alter n-butyrate metabolism. We conclude that some metabolic features of pig colonocytes, such as the capacity to oxidize n-butyrate, resemble those of rat and human colonocytes. Moreover, some characteristics, such as the glycolytic capacity, can be modulated by the level of fiber in the diet.