The rat aldolase B 5' flanking region (nucleotides - 194 to +41) contains sufficient information for liver-specific expression. A detailed investigation of factors binding to the rat aldolase B 5' flanking region has allowed us to identify three distinct factors that filled different sites of this region (A, B, C). The liver-enriched C/EBP or related factors bind to box C, as demonstrated by the specific interaction with bacterially expressed C/EBP protein. Box B bearing the CCAAT sequence binds the ubiquitous factor NFY. Surprisingly, Box A is able to bind two liver enriched factors, namely HNF1 and HNF3. However, in the context of the intact promoter, as shown by footprinting competition experiments, HNF3 binds solely to this sequence. HNF3, but not HNF1 is a transcriptional activator as demonstrated in the in vitro transcription assay.