Previous analyses of the mechanism of the transcriptional induction of the rat haptoglobin (Hp) gene during acute-phase (AP)-reaction have revealed the involvement of several trans -acting nucleoproteins (NPs) in controlling this process. In this study, by using antibodies against C/EBPβ factor in Western immunoblot assay, we found that rat liver trans -acting NPs p35 and p20 are two characteristic C/EBPβ isoforms whose expression is induced under AP-conditions. DNA-binding assays identified the binding sites for these two C/EBPβ proteins in the functionally defined elements A and C of the rat Hp gene and also revealed that they have specific binding affinity towards these elements. Under non-induced conditions, p35 was the only C/EBPβ binding factor; however, upon AP-conditions both, 35kDa- and 20kDa-C/EBPβ binding activities were significantly induced suggesting that these interactions are necessary for the activation of the Hp gene. By in vitro phosphorylation assay and selective proteolysis, we also present evidence that p35 requires phosphorylation for its DNA binding ability. Thus, we conclude that increase in binding of C/EBPβ isoforms during AP-reaction occurs through their upregulation and structural modification.