We have previously found that, in addition to mediating contraction of vascular smooth muscle, activation of alpha1D-adrenergic receptors (AR) induces smooth muscle cell (SMC) hypertrophy. Despite their importance, little is known about how alpha1D-AR expression is regulated. Recently, we demonstrated that platelet-derived growth factor (PDGF)-beta receptor stimulation, but not various other growth factors, inhibits transcription of alpha1D-, but not alpha1A- or alpha1B-ARs, resulting in reduced norepinephrine-mediated SMC growth. To investigate this inhibitory mechanism, herein we cloned and characterized 1.6 kb of the 5'-flanking region of the rat alpha1D-AR gene. Reporter gene transfection assays in rat aorta and vena cava SMCs showed that this 5'-flanking region, which lacks a TATA-box, possesses strong promoter activity. Two transcription initiation sites and their flanking promotor regions were identified, wherein the proximal promotor mediated PDGF-BB inhibition of transcription. Gel mobility shift assays suggested that Sp1 binds constitutively at two consensus sites within the -399 base pair (bp)/-349-bp region of the proximal promotor. This constitutive binding was unaffected by PDGF-BB. In contrast, a flanking motif (-384 bp/-349 bp), possessing putative Sp1/activator protein-2 (AP-2) overlapping binding sites and located upstream of the proximal transcription initiation site, was required for PDGF-BB inhibition of alpha1D transcription. PDGF-BB increased AP-2 binding to the distal AP-2 site in this region in the context of SMCs. Furthermore, overexpression of AP-2 protein, by transgene transfection, dose-dependently inhibited alpha1D-AR activity driven by this motif. Thus, PDGF-BB may increase AP-2 binding within the proximal promoter to cause down-regulation of alpha1D-AR expression in SMCs when PDGF is elevated, such as in the postnatal growing vascular wall and in vascular hypertrophic diseases.