The human alcohol dehydrogenase 5 gene (also known as the formaldehyde dehydrogenase gene, ADH5/FDH) has a GC-rich promoter with many sites at which transcription factors bind. A minimal promoter extending from -34 base pairs (bp) to +61 bp directs high levels of transcription in several different cells, consistent with the ubiquitous expression of the gene. Nearly the entire minimal promoter can be bound by Sp1. We analyzed the transcriptional regulation of ADH5/FDH by members of the Sp1 multigene family. Two core cis-elements (-22 bp to +22 bp) had the highest affinity for Sp1. Mutagenesis revealed that these cis-elements are critical for transcriptional activation. The zinc-finger domains of Sp3 and Sp4 also bind selectively to the core cis-elements. In Drosophila SL2 cells, which lack endogenous Sp1, the minimal promoter cannot drive transcription. Introduction of Sp1 activated transcription over 50-fold, suggesting that Sp1 is critical in the initiation of transcription. Neither Sp3 nor Sp4 was able to activate transcription in those cells, and transcriptional activation by Sp1 was repressed by Sp3 or Sp4. These data suggest that Sp3 and Sp4 can repress transcription by competing with Sp1 for binding to the core cis-elements. The content of Sp1, Sp3, and Sp4 in different cells may be critical factors regulating transcription of the ADH5/FDH gene.