A plasmid containing the CCAAT/enhancer-binding protein (C/EBP alpha) gene transcriptionally controlled by the metallothionein promoter was constructed. The gene was transfected into the human hepatocellular carcinoma cell lines Hep3B and HepG2. When cultured in vitro in the presence of 100 microM ZnSO(4), C/EBP alpha expression caused reversible growth arrest. In soft agar clonogenic assays, C/EBP alpha expression decreased both the colony size and the total number of colonies compared with zinc-free controls. C/EBP alpha expressing cells s.c. implanted in CD-1 nu/nu mice were essentially nontumorigenic, whereas C/EBP alpha tumor cells implanted into immunodeficient SCID mice demonstrated a significantly delayed time of tumor appearance compared with cells transfected with a vector control plasmid. These studies suggest that the expression of endogenous genes normally associated with a quiescent, differentiated state, such as C/EBP alpha, can result in impaired proliferative activity and suppressed tumorigenicity of hepatoma cell lines.