The sequence 5'-GTGGGTGTGGC (G3T) is important for the efficient initiation of transcription from the human ADH2 promoter. We show here that the purified transcription factor Sp1 binds with high affinity to the G3T site of ADH2 (encoding beta beta-alcohol dehydrogenase), even though the G3T sequence does not contain the canonical Sp1-binding site, GGGCGG. Proteins from mouse liver nuclei and purified Sp1 both footprint the same sequence of the ADH2 promoter with similar patterns. UV crosslinking demonstrates that the major G3T-binding protein in the liver extract is similar in size to Sp1. Mouse liver nuclear extract resembles purified Sp1 in its relative binding affinity to a series of oligodeoxyribonucleotides containing either the Sp1-binding site or variants of the G3T sequence. These data indicate that the G3T sequence can interact with Sp1 and that Sp1 may be important in the expression of ADH2. The G3T sequence from the closely related ADH3 gene (encoding gamma gamma-alcohol dehydrogenase) differs from that of ADH2 in the first two nucleotides; it binds both the liver protein and purified Sp1 with lower affinity. This might explain why ADH3 is expressed at lower levels than ADH2 in the liver.