Ammonia decreased metabolism by rat colonic epithelial cells of butyrate and acetate to CO₂ and ketones, but increased oxidation of glucose and glutamine. Ammonia decreased cellular concentrations of oxaloacetate (OAA) for all substrates evaluated. The extent to which butyrate carbon was oxidized to CO₂ after entering the TCA cycle was not significantly influenced by ammonia, suggesting there was no major shift towards efflux of carbon from the TCA cycle. Ammonia reduced entry of butyrate carbon into the TCA, and the proportion of CoA esterified with acetate and butyrate correlated positively with the production of CO₂ and ketone bodies. Also, ammonia reduced oxidation of propionate but had no effect on oxidation of 3-hydroxybutyrate. Inclusion of glucose, lactate or glutamine with butyrate and acetate counteracted the ability of ammonia to decrease their oxidation. In rat colonocytes, it appears that ammonia suppresses SCFA oxidation by inhibiting a step prior to or during their activation. This inhibition is alleviated by glucose and other energy-generating compounds. These results suggest that ammonia may only affect SCFA metabolism in vivo when glucose availability is compromised.