The liver-type and muscle-type isozymes of 6-phosphofructo-2-kinase/fructose- 2,6-bisphosphatase are encoded by the same gene from different promoters. We have found earlier that the liver-type promoter is controlled by ubiquitous and liver-enriched transcription factors and that two cis-acting sequences, called sites III and IV, account for about half of the activity of this promoter (Lemaigre, F. P., Durviaux, S. M., and Rousseau, G. G. (1991) Mol. Cell. Biol. 11, 1099-1106). Site III contains the TGTTTGC sequence, i.e. the hepatocyte nuclear factor 5 motif, hepatocyte nuclear factor 5-TGTTTGC sequence motif, which is known to be functionally important in several liver-specific genes. We now show that site III actually binds the ubiquitous octamer factor 1 and the liver-enriched hepatocyte nuclear factor 3 (HNF-3) in a mutually exclusive way. We also show that site IV binds CCAAT/enhancer-binding protein (C/EBP)-related factors as well as another factor that is liver-specific. This factor is more abundant or has a higher affinity for site IV than the C/EBP-related proteins. Although it can bind to a high affinity HNF-3 site, this factor differs from known members of the HNF-3 family and from other liver-specific transcription factors.