The binding of tritiated etiocholanolone to rat liver and Novikoff hepatoma cells has been investigated. Cytosols obtained from rat liver and Novikoff hepatoma contain high affinity-low capacity binding macromolecules (Kd = 9.6 × 10-10 M for rat liver and 1.1 × 10-9 M for Novikoff hepatoma and the number of binding sites was 64 fmol/mg protein for rat liver and 76 fmol/mg protein for Novikoff hepatoma). Cytosols labelled with [3H]-etiocholanolone had a sedimentation coefficient of 4.6 under low and high salt concentration. Preincubation of rat liver slices and Novikoff hepatoma cells with unlabelled etiocholanolone followed by exchange of nuclear receptor complex with [3H]-etiocholanolone showed the presence of a single peak sedimenting at 4.6 S. Extraction of nuclear receptor peak sedimenting at 4.6 S and identification of the steroid showed that 86% of the radioactivity was associated with etiocholanolone and 14% with 5β-androstane-3α,17β-diol. Competition studies showed that steroids with 5β-H configuration were very effective in displacing etiocholanolone from cytoplasmic receptors while steroids with the 5α-configuration were not. These studies show that etiocholanolone receptor present in mammalian liver is essentially similar to that obtained from chick embryo liver.