Adipocyte differentiation is a complex process triggered and facilitated by transcription factors such as peroxisome proliferator-activated receptor gamma (PPAR gamma) and CCAAT/enhancer-binding protein (C/EBP) alpha. Most about the cascade underlying the differentiation process, especially events in the early stages, remain to be elucidated. Early on in adipocyte differentiation, the C/EBP beta and C/EBP delta genes are rapidly induced to express and later activate PPAR gamma and C/EBP alpha expression. C/EBP beta also plays a crucial role in mitotic clonal expansion (MCE), the approximately two rounds of mitosis which occurs soon after pre-adipocytes are stimulated by differentiation inducers and a necessary step for adipocyte differentiation. However, the effect of C/EBP delta, another member of the C/EBP family, on MCE remains unclear. In the present study, we investigated the role of C/EBP delta in the early stages of adipogenesis. A remarkable induction of C/EBP delta gene expression after the initiation of differentiation was observed not in proliferating preadipocytes, but in growth-arrested, differentiable cells. RNAi-mediated knockdown of C/EBP delta dramatically suppressed cell growth after differentiation was induced, and inhibited conversion into lipid-laden adipocytes. Furthermore, silencing of C/EBP delta impaired the expression of factor for adipocyte differentiation (fad) 49, which is up-regulated and plays a crucial role early in adipogenesis. Taken together, these findings show that C/EBP delta is involved in MCE and gene expression in the early stages of adipocyte differentiation. (C) 2009 Elsevier Masson SAS. All rights reserved.