We have examined the role of protein-protein interactions in modulating the activity of Sp1, a human transcription factor that utilizes glutamine-rich activation domains. These domains may represent a commonly used structural motif, since a nonhomologous glutamine-rich segment from the Drosophila Antennapedia protein is also a potent activator when fused to the Sp1 DNA binding domain. Sp1 is generally considered a proximal promoter factor that can only stimulate transcription when bound close to the initiation site. However, here we present evidence that distally and proximally bound Sp1 can stimulate transcription synergistically. In addition, a DNA binding-deficient mutant of Sp1 that retains glutamine-rich domains can interact with proximally bound Sp1 to superactivate transcription. Glutaraldehyde cross-linking provides direct evidence for an interaction between Sp1 monomers. Thus, Sp1-Sp1 interactions may play an important role in modulating promoter activity.