When small intestinal epithelial cells are incubated with [³H]corticosterone, nuclear binding is displaced neither by aldosterone nor RU-28362, suggesting that [³H]corticosterone is binding to a site distinct from mineralocorticoid receptor and glucocorticoid receptor. Saturation and Scatchard analysis of nuclear [³H]corticosterone binding demonstrate a single saturable binding site with a relatively low affinity (49 nM) and high capacity (5 fmol/µg DNA). Competitive binding assays indicate that this site has a unique steroid binding specificity, which distinguishes it from other steroid receptors. Steroid specificity of nuclear binding mirrors inhibition of the low 11-dehydrogenase activity, suggesting that binding may be to an 11-hydroxysteroid dehydrogenase (11HSD) isoform, although 11HSD1 is not present in small intestinal epithelia and 11HSD2 does not colocalize intracellularly with the binding site. In summary, a nuclear [³H]corticosterone binding site is present in small intestinal epithelia that is distinct from other steroid receptors and shares steroid specificity characteristics with 11HSD2 but is distinguishable from the latter by its distinct intracellular localization.