Expression of platelet thromboxane receptors is transcriptionally increased during megakaryocytic differentiation stimulated by phorbol 12-myristate 13- acetate (PMA). We previously cloned and characterized the promoter region of the human thromboxane receptor gene and localized PMA-responsive elements to a region between 1.84 and 1.95 kilobase pairs (kb) 5' of the transcription initiation site (D'Angelo, D. D., Davis, M. G., Houser, W. A., Eubank, J. J., Ritchie, M. E., and Dorn, G. W., II (1995) Circ. Res. 77, 466-474). Herein we report the localization of the PMA response element to a 14-nucleotide C-rich sequence, flanked by an octanucleotide inverted repeat, located -1.938 to - 1.925 kb 5' of the transcription start site of this gene. We further identify the PMA-responsive enhancer factor that binds to this C-rich sequence as Sp1. Heterologous thromboxane receptor gene promoter/thymidilate kinase reporter constructs transfected into K562 cells exhibited PMA responsiveness when the C-rich element was included with additional 3' sequence from -1.924 to -1.84 kb. However, mutations of the C-rich element that disrupted a GC box located on the inverse strand eliminated PMA responsiveness and, in gel mobility shift assays, eliminated binding of Sp1. PMA treatment of K562 cells significantly increased, by 5-fold, Sp1 binding to the C-rich element and increased both phosphorylated and nonphosphorylated Sp1 protein levels by 2-fold. Furthermore, pMA treatment transiently increased Sp1 mRNA levels prior to increasing thromboxane receptor mRNA, suggesting that up-regulation of Sp1 contributes to up-regulation of thromboxane receptors. Finally, we have detected an unidentified K562 nuclear protein that binds specifically to the sense strand of the C-rich sequence overlapping the Sp1 binding site and that, by stabilizing a double stem-loop conformation of this DNA segment, may also play a role in Sp1 regulation of this gene. These studies are the first to describe regulatory and regulated roles for Sp1 in PMA-responsive gene expression and suggest that modulation of Sp1 levels controls thromboxane receptor expression during megakaryocytic differentiation.