The megakaryocyte-specific integrin, alpha IIb, is the alpha-subunit of the alpha IIb/beta 3 complex found on the surface of platelets. This complex is a receptor for fibrinogen and other ligands when platelets are activated. Because the alpha IIb gene is specifically expressed in megakaryocytes, the 5′-flanking region was studied as a potential model for megakaryocyte-specific gene expression. Previous studies have defined some of the important regulatory elements in the 5′-flanking region of this gene. The present studies focus on the issue of the molecular basis by which this TATA-less gene is properly transcribed. A GA-rich region centered 14 bp upstream from the transcriptional start site appears to be a nonconsensus Sp1-binding site. Binding to this site is of low affinity, but is markedly improved by interaction with protein(s) binding at an Ets-consensus site approximately 20 bp further upstream. Mutation of the Ets site greatly reduces the ability of Sp1 to bind to its site. Trans-acting nuclear factors binding to and interaction of the proteins at these two sites have direct effects on the observed promoter activity in primary megakaryocyte transient expression studies. These studies provide further evidence of the role of interactions between Ets-like proteins and Sp1 in transcriptional activation when a TATA box is not present in the promoter region of a gene. Based on the presented studies and previous results, a model is proposed for the regulation of expression of the alpha IIb gene.