The colonic mucosa may be especially vulnerable during starvation and malnutrition, as luminal nutrients make the greatest contribution to its energy production. To investigate possible metabolic changes in the colonic mucosa during nutrient restriction, we studied substrate utilization by colonocytes isolated from three groups of 6-wk-old rats: control, fasted (72 h), and chronically malnourished animals. Isolated colonocytes were incubated with nonlabeled and 14C-labeled substrates (glucose, glutamine, n-butyrate, or beta-hydroxybutyrate). Substrate oxidation and net increase of intermediary metabolites were reduced in fasted and malnourished animals. The effect of fasting on substrate oxidation was greater than that of chronic malnutrition for all substrates tested except n-butyrate. The total ketone body concentrations and beta-hydroxybutyrate to acetoacetate ratios were higher in the fasted and malnourished groups than in controls. The findings suggest that the colonic mucosa responds to nutrient deprivation by a general reduction of oxidative metabolism that is associated with an altered redox state.