The αIIb/β3-integrin receptor is present at high levels only in megakaryocytes and platelets. Its presence on platelets is critical for hemostasis. The tissue-specific nature of this receptor's expression is secondary to the restricted expression of αIIb, and studies of the αIIb proximal promoter have served as a model of a megakaryocyte-specific promoter. We have examined the αIIb gene locus for distal regulatory elements. Sequence comparison between the human (h) and murine (m) αIIb loci revealed high levels of conservation at intergenic regions both 5′ and 3′ to the αIIb gene. Additionally, deoxyribonuclease (DNase) I sensitivity mapping defined tissue-specific hypersensitive (HS) sites that coincide, in part, with these conserved regions. Transgenic mice containing various lengths of the hαIIb gene locus, which included or excluded the various conserved/HS regions, demonstrated that the proximal promoter was sufficient for tissue specificity, but that a region 2.5 to 7.1 kb upstream of the hαIIb gene was necessary for consistent expression. Another region 2.2 to 7.4 kb downstream of the gene enhanced expression 1000-fold and led to levels of hαIIb mRNA that were about 30% of the native mαIIb mRNA level. These constructs also resulted in detectable hαIIb/mβ3 on the platelet surface. This work not only confirms the importance of the proximal promoter of the αIIb gene for tissue specificity, but also characterizes the distal organization of the αIIb gene locus and provides an initial localization of 2 important regulatory regions needed for the expression of the αIIb gene at high levels during megakaryopoiesis.