Recent studies reveal important and distinct roles for cardiac ␣ 1a adrenergic receptors (␣ 1a ARs). Surprisingly, given their importance in myocardial ischemia/reperfusion, hypoxia, and hypertrophy as well as frequent use of rat cardiomyocyte model systems, the rat ␣ 1a AR gene promoter has never been characterized. Therefore, we isolated 3.9 kb of rat ␣ 1a AR 5-untranslated region and 5-regulatory sequences and identified multiple transcription initiation sites. One proximal (P1) and several clustered upstream distal promoters (P2, P3, and P4) were delineated. Sequences surrounding both proximal and distal promoters lack typical TATA or CCAAT boxes but contain cis-elements for multiple myocardium-relevant nuclear regulators including Sp1, GATA, and CREB, findings consistent with enhanced cardiac basal ␣ 1a AR expression seen in Northern blots and reporter constructs. Promoter analysis using deletion reporter constructs reveals, in addition to a powerful upstream enhancer, a key region (-558/؊542) important in regulating all ␣ 1a AR promoters with hypoxic stress. Gel shift analysis of this 14-bp region confirms a hypoxia-induced shift independent of direct hypoxia-inducible factor binding. Mutational analysis of this sequence identifies a novel 9-bp hypoxia response element, the loss of which severely attenuates hypoxia-mediated repression of ␣ 1a AR transcription. These findings for the ␣ 1a gene should facilitate elucidation of ␣ 1 AR-mediated mechanisms involved in distinct myocardial pathologies.␣ 1 ARs 1 are members of the larger family of G protein-coupled receptors that mediate sympathetic nervous system responses such as smooth muscle contraction (1) and increased myocardial contractility (2, 3); these responses occur predominantly via activation of phospholipase C-␤, resulting in the stimulation of both protein kinase C and inositol trisphosphate pathways (4). cDNAs encoding three ␣ 1 AR subtypes (␣ 1a , ␣ 1b , and ␣ 1d ) have been cloned and pharmacologically characterized in several expression systems, leading to the surprising finding that ␣ 1 AR subtype tissue distribution is species-dependent, with expression regulated at both gene and protein levels (4). To gain insight into transcriptional pathways governing ␣ 1 AR expression, several laboratories have initiated cloning and characterization of ␣ 1 AR subtype regulatory regions from different species, including the ␣ 1a AR (human (5), mouse (6)), ␣ 1b AR (mouse (7), rat (8), human (9)), and ␣ 1d AR (rat (10)).Recent studies reveal an important and distinct role for ␣ 1a ARs in the heart, including enhanced contractility (2, 3) and hypertrophy (11)(12)(13)(14). Modulation of ␣ 1a AR expression occurs during pathological states, such as myocardial hypertrophy (13, 14) and hypoxia (15), the latter usually secondary to ischemic injury. In hypertrophic models, classically performed in cultured neonatal rat cardiomyocytes, direct chronic ␣ 1a AR stimulation results in increased transcription of the ␣ 1a AR gene itself, providing a pathway for overall s...