The possible induction of renal citrate synthase (E.C. 126.96.36.199), by aldosterone was evaluated in the adrenalectomized rat. Three hours after administration of aldosterone (0.8 μg/100 g body wt), renal cortical and medullary citrate synthase activity was significantly increased as reported previously by Kinne and Kirsten (Kinne, R., Kirsten, R. 1968. Pfleugers Arch. 300:244). In contrast, no change in this activity was detected in the renal papilla or the liver, under the same conditions. Kinetic analysis revealed that injection of aldosterone had no effect on the Kms for acetyl-CoA and oxalacetate but augmented Vmax of renal medullary citrate synthase activity by 40%. The aldosterone-dependent increase in medullary citrate synthase activity was proportionate to the associated increase in the quantity of antiserum (specific for citrate synthase) required for half-maximal immuno-precipitation. The possibility that aldosterone induced the synthesis of citrate synthase was evaluated in two sets of experiments. In the first set, adrenalectomized rats were injected intraperitoneally with either aldosterone (0.8 μg/100 g body wt) or the diluent, and simultaneously with3H or35S methionine (500 μCi/rat). The isotopes were reversed in about half of the experiments. Three hours after the injection, renal citrate synthase was isolated by ATP-sepharose column chromatography and immuno-precipitation with the specific antiserum. Aldosterone augmented methionine incorporation into renal citrate synthase by 55% but had no effect on incorporation into the hepatic enzyme. In the second set, adrenalectomized rats were injected with either aldosterone (0.8 μg/100 g body wt) or the diluent, the kidneys were removed 1 hr later and medullary slices were incubated in either3H-or35S-methionine at 20° for 2 hr. Mitochondrial citrate synthase was isolated either by ATP-sepharose column chromatography and immuno-precipitation, or by polyacrylamide gel electrophoresis. Aldosterone increased methionine incorporation into the immuno-precipitates by 30% and into the enzyme peak resolved by polyacrylamide gel electrophoresis by 43%. The latter increase was eliminated by prior administration of either actinomycin D (70-80 μg/100 g body wt) or spirolactone (SC-26304) (80 μg/100 g body wt). An equimolar dose of dexamethasone (0.8 μg/100 g body wt) had no effect on the isotope ratio associated with citrate synthase activity in the polyacrylamide gels.