C/EBPbeta is one of the key transcription factors responsible for the induction of a wide array of genes. Like many proto-oncogenes and transcription factors, transcription of C/EBPbeta gene can be induced by multiple extracellular signals. Using nuclear extracts from lipopolysaccharide (LPS)-stimulated mouse liver, five trans-acting factor-binding motifs, URE1 (-376 to -352), URE2 (-253 to -223), URE3 (-220 to -190), URE4 (-123 to -103), and URE5 (-72 to -45) were identified by DNAse I footprinting assays. Competition and supershift analysis of the complexes formed at the URE2 and URE4 indicated that they contain CREB/ATF and AP-1 family factors. Furthermore, recombinant ATF2 and c-Jun proteins from mammalian and bacterial cells can bind to URE2 and URE4 but not URE1. Cotransfection experiments showed that ATF2 and c-Jun activate the C/EBPbeta gene expression cooperatively through URE2 and URE4, and this activation was greatly increased under the treatment of low concentration of anisomycin. During acute phase response, the phosphorylation of c-Jun and ATF2 was found to correlate with C/EBPbeta gene expression. Taken together, our results provide the evidences that both c-Jun and ATF2 are the regulators of C/EBPbeta gene.