Knockout of C/EBPα causes a severe loss of liver function and, subsequently, neonatal lethality in mice. By using a gene replacement approach, we generated a new C/EBPα-null mouse strain in which C/EBPβ, in addition to its own expression, substituted for C/EBPα expression in tissues. The homozygous mutant mice C/ebp αβ/β are viable and fertile and show none of the overt liver abnormalities found in the previous C/EBPα-null mouse line. Levels of hepatic PEPCK mRNA are not different between C/ebp αβ/β and wild-type mice. However, despite their normal growth rate, C/ebp αβ/β mice have markedly reduced fat storage in their white adipose tissue (WAT). Expression of two adipocyte-specific factors, adipsin and leptin, is significantly reduced in the WAT of C/ebp αβ/βmice. In addition, expression of the non-adipocyte-specific genes for transferrin and cysteine dioxygenase is reduced in WAT but not in liver. Our study demonstrates that when expressed from the C/ebp α gene locus, C/EBPβ can act for C/EBPα to maintain liver functions during development. Moreover, our studies with the C/ebp αβ/β mice provide new insights into the nonredundant functions of C/EBPα and C/EBPβ on gene regulation in WAT.