In an attempt to identify proteins that may regulate α ₁ -fetoprotein (AFP) gene expression, we screened a cDNA expression library from neonatal rat liver with two essential cis-elements of the AFP promoter and enhancer. We isolated two cDNAs which were found to correspond to leucine zipper proteins of the CC-AAT/enhancer binding protein (C/EBP) family: C/EBPβ and C/EBPγ. The three related proteins C/EBP α, β and γ bind with indistinguishable specificity to multiple DNA sites in the promoter and the enhancer of the AFP gene. In addition, C/EBPβ and C/EBPγ readily heterodimerize with each other as well as with C/EBPα. The mRNAs coding for C/EBPβ and C/EBPγ are expressed in a wider variety of rat tissues than C/EBPα mRNA, including yolk sac and fetal liver. The steady-state levels of C/EBP α, β and γ mRNAs increase during liver development, in parallel with their respective gene transcriptional rates. The high levels of C/EBP β and γ mRNAs in rat yolk sac and fetal liver, where C/EBPα is poorly expressed, suggest that C/EBP β and/or γ could be preponderant or early regulators of the AFP gene in these tissues.