The Sp1 protein activates transcription from many eukaryotic promoters. Sp1 can act in vivo from enhancer sites that are distal to the promoter and exhibit synergistic interaction with promoter-proximal binding sites. To investigate possible protein-protein interactions between DNA-bound Sp1 molecules, we have used electron microscopy to visualize the DNA-protein complexes. At the SV40 promoter, we observed the expected localized interaction at the Sp1 sites; in addition, we found that DNA-bound Sp1 served to associate two or more DNA molecules. At a modified thymidine kinase promoter, we observed a localized interaction at each of two binding locations that were separated by 1.8 kbp; in addition, we noted a substantial fraction of DNA molecules in which the distant binding regions were joined by a DNA loop. As judged by studies with mutant Sp1 proteins, the distant interactions depended on the glutamine-rich regions of Sp1 required for transcriptional activation. We conclude that DNA-bound Sp1 can self-associate, bringing together distant DNA segments. From the correlation between DNA looping in vitro and synergistic activation of the modified thymidine kinase promoter shown previously in vivo, we suggest that Sp1 exerts its transcriptional synergism by a direct protein-protein association that loops the intervening DNA. Our experiments support the DNA-looping model for the function of transcriptional enhancers.