The binding of metabolites of vitamin D and their analogs to the 3.7S chick intestinal cytosol receptor protein has been specifically studied by competitive binding techniques and polyethylene glycol precipitation of the complex. The structural requirements for the interaction between the vitamin D molecule and the receptor could be assessed without the nuclear chromatin binding step. These measurements have shown that 1,25-dihydroxyvitamin D3 and 1,25-dihydroxyvitamin D2 are equally competitive and are the most active. Of the structural features of the compounds, the 1 alpha-hydroxyl is most important followed by the 25-hydroxyl and the 3 beta-hydroxyl. The addition of a second hydroxyl near carbon 25 markedly reduces binding whether on the 26 carbon or the 24 carbon. A hydroxyl on C-24 could substitute to some degree for the 25-hydroxyl inasmuch as 24-hydroxyvitamin D3 was much more effective than vitamin D3 but less effective than 25-hydroxyvitamin D3. In general the patterns of binding affinities correlated well with the biological activity of the various analogs strongly supporting a physiological role for the 1,25-dihydroxyvitamin D3 binding protein. It also suggests that of the two-step receptor mechanism, the structural specificity is located in the initial interaction of the 1,25-dihydroxyvitamin D3 and the cytosol receptor.