A 5β-H steroid binding component using etiocholanolone has been identified in chick embryo liver which exhibits characteristics of a specific receptor. Kinetic analysis revealed the presence of specific binding sites with a Kd of 8.9 × 10-10 M and a concentration of binding sites of 59 fmol/mg protein. The nuclear and cytosol macromolecule has a sedimentation coefficient of 4.6 under low and high salt conditions. Following in vivo injection of etiocholanolone to chick embryos or in vitro preincubation of chick embryo tissue slices at 30°C the cytosol binding protein was markedly reduced while the nuclear binding protein increased. However, etiocholanolone preincubation of tissue slices at 0°C resulted in no receptor translocation. Competition studies using a 200-fold excess of various unlabelled steroids demonstrated greatest inhibition with steroids having the 5β-H configuration. These data offer strong evidence for the presence of a 5β-H steroid receptor in chick embryo liver.