Studies on the effects of pretreatment with aldosterone on the incorporation of3H leucine or3H methionine into proteins in renal slices were carried out in Joklik-modified minimal essential medium. Administration of aldosterone (2 μg/100 g body wt) to adrenalectomized rats increased3H leucine incorporation into trichloroacetic acid insoluble fractions of crude homogenates of cortical slices by 15.5±0.4% and of medullary slices by 53.5±1.3%. No increase in isotope incorporation was observed in slices of renal papilla or spleen prepared from the same rats. Aldosterone had no effect on the3H-leucine content of the trichloroacetic acid-soluble fractions of all three renal zones and the spleen. The dose of aldosterone that elicited a half-maximal increase in3H-methionine incorporation into proteins of renal medullary slices (0.45 μg of aldosterone/100 g body wt) was indistinguishable from that needed to elicit a halfmaximal increase in the urinary K+/Na+ ratio (0.35 μg of aldosterone/100 g body wt). Dexamethasone, a potent glucocorticoid, at a dose of 0.8 μg/100 g body wt did not augment3H-leucine incorporation into renal medullary proteins but was effective at 8 μg/100 g body wt. Spirolactone (SC-26304), a potent anti-mineralocorticoid, abolished the effect of aldosterone on amino acid incorporation into medullary proteins when administered at a 100-fold higher dosage [i.e., 80 μg vs. 0.8 μg (per 100 g body wt)]. These results imply that the action of aldosterone on amino acid incorporation is mediated by the mineralocorticoid rather than the glucocorticoid pathway, presumably the mineralocorticoid receptors. Moreover, pretreatment of the rats with actinomycin D (70-80 μg/100 g body wt) erased the effect of aldosterone (0.8 μg/100 g body wt) on amino acid incorporation into medullary proteins. In paired experiments with3H and35S methionine, aldosterone (0.8 μg/100 g body wt) increased methionine incorporation into trichloroacetic acid precipitable proteins of subcellular fractions of the renal medulla. The effect of aldosterone on incorporation of methionine into medullary cytosol proteins was analyzed further by polyacrylamide gel electrophoresis at pH 8.3 in tris-glycine buffer. The gel profiles indicate that aldosterone significantly increased methionine incorporation into at least one protein (independent of the isotope) with a molecular weight of ∼31,000. This increase was inhibited by either pretreatment of the rat with actinomycin D (70-80 μg/100 g body wt or SC-26304 (80 μg/100 g body wt). Dexamethasone (0.8 μg/100 g body wt) did not increase incorporation of methionine into the medullary cytosol proteins resolved by polyacrylamide gel electrophoresis.