The transcription factor CCAAT/enhancer-binding protein (C/EBP) was found to selectively trans-activate one member of the human class I alcohol dehydrogenase (ADH) gene family. A comparison of the promoters for the three human class I ADH genes ADH1, ADH2, and ADH3 indicated a very similar pattern of binding sites (sites A-F) for rat liver nuclear proteins located between -10 and -210 base pairs (bp). In all three promoters site A consisted of two binding sites for the transcription factor C/EBP closely flanking both sides of the TATA box, but C/EBP bound with much greater affinity to site A of ADH2. C/EBP also bound at two locations which coincide with site D (-120 bp) and site E (-160 bp) of all three promoters. Cotransfection studies of human hepatoma cells using ADH-cat fusions and a C/EBP expression plasmid indicated that the human ADH2 promoter responded well to C/EBP trans-activation whereas the human ADH1 and ADH3 promoters, which bind C/EBP weakly, responded poorly. Individual mutations in several ADH2 nuclear factor-binding sites allowed the identification of four functional C/EBP-binding sites, i.e. two in site A as well as one each in sites D and E. Also, the ADH2 TATA box was found to be dispensable for C/EBP induction. Compared to ADH2 and ADH3, site A in ADH1 contains four extra base pairs between the two C/EBP motifs, and deletion of these nucleotides increased the C/EBP responsiveness of ADH1 presumably by changing the spacing of the two C/EBP motifs. Thus, sequence divergence of human class I ADH gene family members has led to forms which vary in their responsiveness to C/EBP. We suggest that C/EBP contributes to liver-specific expression of the human class I ADH gene family by selectively inducing the ADH2 gene via a TATA-independent mechanism during liver development.