Abstract A rapid and sensitive method for evaluating the specific internalization of [ 3H]1,25-(OH) 2-D 3 by intact, cultured intestinal epithelial (IEC) cells was developed. The uptake of hormone was determined by incubation of suspensions of IEC cells in medium containing [ 3H]1,25-(OH) 2-D 3 and 1% fetal bovine serum. Saturation of the high affinity intracellular receptor with 1,25-(OH) 2-D 3 was reflected in an analysis of the saturable uptake of [ 3H]1,25-(OH) 2-D 3 the apparent dissociation constant for labelled hormone was 0.64 nM which is in the range of that reported for hormone binding to receptor extracted from known target tissues. The uptake of [ 3H]1,25-(OH) 2-D 3 was dependent on both the cellular content of receptor and the availability of unbound hormone in the extracellular medium. The latter phenomenon was demonstrated in competitive internalization studies with 25-OH-D 33 and 24,25-(OH) 2-D 3. At extracellular concentrations of 25-OH-D 3 and 24,25-(OH) 2-D 3 ranging from 10 −8 to 10 −7M there was competitive inhibition of [ 3H]1,25-(OH) 2-D 3 uptake by IEC cells. However, when cells were exposed to a higher concentration (10 −6M) of these metabolites, label uptake paradoxically increased owing to probable saturation of the sterol binding sites on the vitamin D binding protein present in the serum-supplemented incubation medium.