Abstract Gene 33, a rat gene transcriptionally enhanced by glucocorticoids, insulin, or cyclic AMP, was isolated from a library of rat genomic DNA and characterized by sequence comparison to a full-length cDNA. The structural gene spans 13 500 bp encoding 2970 bp of exon sequences interrupted by three introns of about 9600, 101 and 811 bp, respectively. Exons (5' to 3') are 198, 194, 77 and 2501 bp in length; the first of these initiates at the transcriptional start point determined by S1 nuclease mapping. The 5'-flanking DNA contains several putative transcriptional control elements including TATA and CAAT boxes and a binding site for the Sp1 transcription factor in the usual locations proximal to the start point. Sequences resembling known glucocorticoid and cyclic AMP regulatory elements are also found upstream. A chimeric plasmid was constructed containing putative gene 33 regulatory elements fused to the Escherichia coli gene cat, encoding the enzyme chloramphenicol acetyltransferase, and transfected into cultured fibroblasts. Transient expression assays established that this gene 33 DNA is effective in promoting transcription.