The highly conserved ornithine decarboxylase (E.C.4.1.1.17) promoter contains multiple binding sites for Sp1 within the first 400 bp of the cap site. Therefore the ability of individual Sp1 elements to confer transactivation alone or in combination was tested. We show that different Sp1 sites vary in their affinity for Sp1 and that increasing affinity correlates with enhancer activity. In addition, several adjacent Sp1 sites synergistically enhanced promoter activity. Thus, the strength of promoter transactivation correlated with both the number of GC-rich elements and their affinity for Sp1 protein.