Colonic metabolism may be affected by dietary fiber and short-chain fatty acids, the products of fiber fermentation. The aim of this study was to assess the effects of fiber supplementation (150 g/kg diet) on dynamic measurements of metabolism in isolated rat colonic epithelial cells. Additionally, we investigated the effect of in vitro short-chain fatty acid and glutamine concentrations and media osmolarity on oxygen uptake, protein synthesis, cell proliferation and anaplerotic flux. Colonocyte oxygen consumption did not differ due to fiber supplementation or the inclusion of short -chain fatty acids in incubation media. Cell proliferation (3H-thymidine uptake) was increased by fiber consumption (P </= 0.05) but was decreased by supplementing short-chain fatty acids in vitro (P </= 0.05), independent of osmolarity. Protein synthesis (3H-phenylalanine incorporation) was unaffected by fiber supplementation but was decreased when short-chain fatty acids were present in incubation media (P </= 0.05). However, this decrease was also induced by in vitro supplementation with isoosmotic NaCl concentrations. These data show that although fiber supplementation increases in vitro colonocyte proliferation, the unchanged oxygen uptake rate indicates that there was no concurrent increase in energy expenditure.