The transcription factor Sp1 has been implicated in regulation of the expression of the murine CTP:phosphocholine cytidylyltransferase alpha (CTalpha) gene, Ctpct (M. Bakovic, K. Waite, W. Tang, I. Tabas, and D. E. Vance. 1999. Biochim. Biophys. Acta. 1438: 147;-165). We have utilized transient transfections, mutation analysis, electromobility gel-shifts, and immunoblot analysis to test the hypothesis that expression of the CTalpha gene is controlled in part by the binding of three trans-acting nuclear factors, Sp1, Sp2, and Sp3. Sp1 and Sp3 activate CTalpha gene transcription through sequence specific binding within three promoter domains. In Sp1-mediated transcription, Sp3 acts as an activator in a dose-dependent manner and vice versa. Sp2 represses Sp1- and Sp3-driven transcription in Drosophila SL2 cells, but stimulates transcription in C3H10T1/2 mammalian cells. Our results suggest that the predominant action of Sp proteins is a direct function of local organization of three cis-acting elements in the regions A (-31/-9), B (-88/-50), and C (-148/-128). The ability of distal C (-148/-128) and proximal A (-31/-9) regions to activate or repress transcription depends upon the cellular background. The multiple binding elements at position B (-88/-50) confer a positive regulation independent of the cell context. However, the effectiveness of Sp proteins at this site is strongly governed by neighboring sites A and C. The results suggest that the level of expression of the CTalpha gene will depend on the cell type, the availability of Sp proteins, and the structure and organization of three cis-acting elements.