To investigate the role of C/EBP family members during adipocyte differentiation in vivo, we have generated mice lacking the C/EBPbeta and/or C/EBPdelta by gene targeting. Approximately 85% of C/EBPbeta(-/-).delta(-/-) mice died at the early neonatal stage. By 20 h after birth, brown adipose tissue of the interscapular region in wild-type mice contained many lipid droplets, whereas C/EBPbeta(-/-).delta(-/-) mice did not accumulate droplets. In addition, the epidydimal fat pad weight of surviving adult C/EBPbeta(-/-).delta(-/-) mice was significantly reduced compared with wild-type mice. However, these adipose tissues in C/EBPbeta(-/-).delta(-/-) mice exhibit normal expression of C/EBPalpha and PPARgamma, despite impaired adipogenesis. These results demonstrated that C/EBPbeta and C/EBPdelta have a synergistic role in terminal adipocyte differentiation in vivo. The induction of C/EBPalpha and PPARgamma does not always require C/EBPbeta and C/EBPdelta, but co-expression of C/EBPalpha and PPARgamma is not sufficient for complete adipocyte differentiation in the absence of C/EBPbeta and C/EBPdelta.