Unlike the β2AR, the β1AR gene promoter has been less well characterized. The lack of hormone responsiveness of the β1AR in the fetus and upregulation by steroids in the newborn and adults may represent a unique transcription mechanism of this physiologically critical receptor. With transient transfection of progressively deleted ovine β1AR gene promoter, we have identified the promoter region conferring glucocorticoid responsiveness. Gel retardation assay using this region of the promoter showed a cell type specific effect where band shifts were observed in SK-N-MC but not in C6 cell nuclear extracts. We used DNase I footprinting to confirm that nucleotides -1260 ≈ -1247, upstream of a GRE ½ site, interact with SK-N-MC cell nuclear proteins. The wild type nucleotides and its corresponding mutant sequences were subcloned into the pGL2-C vector for further studies. While the wild type sequence showed different band shifts from that of mutant sequence in gel retardation assay, no supershift was observed when a glucocorticoid receptor antibody was included in the assay mixture. Further, transient transfection indicated a significant increase in transcriptional activity induced by the wild type sequences. We conclude that nucleotides-1260 ≈ -1247, although conferring cell-type specific glucocorticoid responsiveness, do not appear to interact with GR directly. We speculate that this region in the ovine β1AR promoter may be responsible for its unique transcription regulation.