Betamethasone (BM) receptor cncentrations in the liver cytosol from adrenalectomized rats treated with 0.2 and 2 mg BM were measured and correlated with BM concentrations in the serum and liver. The BM levels in the serum, liver cytosol and nuclei changed in a parallel fashion. One hr after BM administration, serum BM reached a peak, then decreased gradually and was undetectable at 24-48 hr. The peak levels of BM in the serum, liver cytosol and nuclei from 0.2 mg BM treated rats were 880.0±96.0 ng/ml, 32.5±8.1 ng/mg protein and 9.6±2.4 ng/mg DNA or 12.1 ±2.6 ng/g wet liver. Those from 2 mg BM treated rats were 1540.0±942.0 ng/ml, 47.4±38.8 ng/mg protein and 14.2 ± 3.7 ng/mg DNA or 16.6 ±5.6 ng/g wet liver. In the liver cytosol, there are two types of binding sites for BM, one with high affinity (Kd=6.0×10-9 mol/liter) and low capacity (6.6×10-13 mol/mg protein or 13.0±5.2 ng/g wet liver) and one with low affinity (Kd>10-7 mol/liter) and high capacity (>10-11 mol/mg protein). The peak levels of BM in the liver nuclei from 0.2 and 2 mg BM treated rats were close to the binding capacity of high affinity binding site in cytosol (12.1 and 16.6 vs. 13.0 ng/g wet liver). [3H]BM binding to the liver cytosol from both 0.2 and 2 mg BM treated rats was lost completely at 1 to 6 hr, and recovered at 24 hr in the 0.2 mg BM treated rats and at 48 hr in the 2 mg BM treated rats. Thus, the cytosol and nuclear levels of BM are reciprocally related to [3H] BM binding capacity in the liver cytosol, and the fall of [3H] BM binding to the cytosol is accompanied by the appearence of BM in the nuclei. Therefore, our in vivo study suggested that almost all of high affinity receptor bound BM is transferred rapidly to nuclei and remains there in the presence of a sufficient amount of BM in cytoplasm, and thereafter the receptor is released from nuclei to cytoplasm.