To study the mechanism underlying glucocorticoid regulation of the β₁-adrenergic receptor (β₁AR), we identified a 43-bp region (−1274 to −1232 from the translation start site) that contains a novel glucocorticoid regulatory unit (GRU) that confers glucocorticoid responsiveness. The sequence encompassing the GRU is ^5′TAATTA^3′, which is a core-binding motif for the homeodomain proteins; an E-box (^5′CACGTG^3′) binding site for the Myc/Max family proteins, and an overlapping glucocorticoid response element half-site (^5′TGTTCT^3′). We showed that the half-site is critical for GRU-protein interactions, which also require binding of proteins to the E-box and the homeodomain region. Expression of proteins binding to the GRU was shown to be developmentally regulated, being high in embryonic hearts, reduced in newborn hearts, and undetectable in adult hearts. Overexpression of c-myc antisense significantly reduced glucocorticoid responsiveness of the β₁AR gene. We further demonstrated that transcriptional regulation of the β₁AR gene is closely related to that of the c-myc gene and that the β₁AR may be a potential target of c-myc. We conclude that the ovine β₁AR gene contains a novel, functional GRU and that the nuclear factors that transactivate through this element may have important developmental implications. (J Allergy Clin Immunol 2002;110:S247-54.)