Renal cortical slices from acidotic dogs (NH4CL) were incubated at pH 7.0 with L-glutamine (1 and 5 mM) or L-glutamate (5 mM) with or without DL-beta-hydroxyburyrate 1 mM, acetoacetate 0.5 mM, as well as oleate, palmitate, octanoate, and crotonate 0.5 mM and in the presence or absence of fluoroacetate 0.05 mM. Fluoroacetate alone increased glutamine or glutamate uptake and ammoniagenesis whereas gluconeogenesis rose only when glutamine 5 mM or glutamate were used. Alanine production also rose by 30% when glutamine but not glutamate was used as substrate in the presence of fluoroacetate. Efficient blocking of the Krebs cycle at the aconitase level by fluoroacetate was evidenced by the release of citrate in the incubation medium and by a slight but significant decrease in oxygen consumption (10-20%). The marked decrease in glutamine uptake, ammoniagenesis, and gluconeogenesis induced by ketone bodies and fatty acids was completely corrected by addition of fluoroacetate. The present studies demonstrate that ketone bodies and fatty acids inhibit renal ammoniagenesis and gluconeogenesis in vitro through their oxidation in the mitochondria. They also suggest that direct transamination of glutamine into alanine (glutaminase II pathway) may be significant when oxidation of pyruvate is inhibited by fluoroacetate.