Calbindin D28K (formerly Vitamin D-Dependent Calcium Binding Protein) is a protein induced by 1,25-dihydroxycholecalciferol in several chicken tissues (1). A chicken genomic DNA library was screened with a synthetic oligonucleotide representing the sequence of Calbindin D28K cDNA (2,3) from nt 146 to nt 176. The positive clone CBA1 extends the 5'-end of the first exon by 451 bp. Panel a, in the figure, shows the sequence of a BamHI-SacII restriction fragment with coordinates -451 +50. A possible "TATA" box and the C residue appearing as first nt in the cDNA sequence (3) are underlined. The BamHI-SacII fragment was subcloned 5' to the CAT gene of pUCCAT (4). In panel b the result is shown of a CAT assay on mouse fibrohlasts 3T6 transiently transfected with pUCCAT (A), pUCCAT containing the BamHI-SacII fragment in the correct (B) or opposite IS.) orientation or the SV40 promoter (D). E and F respectively indicate C-chloramphenicol and its acetyl derivatives generated by purified CAT. The expression of CAT appears to be constitutive since the enzyme activity is not influenced by the presence (+) or absence (-) of 1,25-dihydroxycholecalciferol in the culture medium.