Promoters of growth and cell cycle regulated genes frequently carry binding sites for transcription factors of the E2F and Sp1 families. We have demonstrated recently that direct interaction between Sp1 and a subgroup of the E2F factors is essential for the regulation of certain promoters. We show here that the amino acids necessary for this interaction in both cases are located within the DNA binding domain. This is in line with the assumption, that the interaction between E2F and Sp-factors contributes to promoter-specificity. Cyclin A, which binds to E2F-1 in close vicinity to Sp1 does not interfere with this interaction. Moreover we have investigated the ability of other members of the Sp1 family to interact with E2F-1 and to regulate the activity of the E2F and Sp1 dependent murine thymidine kinase promoter. All four factors of the Sp1 family are able to bind E2F-1 in co-immunoprecipitation and GST-pull down experiments. Mobility shift assays with oligonucleotides comprising the Sp1, or both the Sp1 and the E2F binding site suggest that Sp1 and Sp3 supply most if not all activity binding to the GC-box of the thymidine kinase promoter in murine fibroblasts. Reporter gene assays in Drosophila melanogaster SL2 cells and murine fibroblast 3T6 cells demonstrate that the thymidine kinase promoter is activated strongly by Sp1 and Sp3, weakly by Sp4, and not at all by Sp2. Co-expression of E2F-1 results in synergistic activation in 3T6 but not in SL2 cells.