p38 beta mitogen-activated protein kinase activity is required for the differentiation of 3T3-L1 fibroblasts into adipocytes. Activating transcription factor-2 (ATF-S) is efficiently phosphorylated and activated by p38 beta kinase. These findings led us to examine a regulatory role of ATF-S in adipocyte differentiation. The induction of ATF-8 protein precedes the expression of the transcription factors, peroxisome proliferator-activated receptor (PPAR) gamma and CCAAT/enhancer-binding protein (C/EBP) alpha. Consistent with early activation of p38 beta kinase, the phosphorylation of ATF-2 was also detected in early stage of adipocyte differentiation. ATE-2 regulated gene transcription of PPAR gamma, which was synergistically enhanced by p38 beta kinase and C/EBP beta proteins expression. Ectopic expression of ATF-8 in 3T3-L1 cells induced the endogenous PPAR gamma protein levels. These results suggest that ATF-2 plays a role in a primary regulator of adipocyte differentiation with C/EBP beta through promoting adipogenesis-inducing transcription factors including PPAR gamma and becomes associated earlier in the differentiation program as mitotic clonal expansion proceeds and the cells become initially differentiated.