C/EBPδ (CCAAT/enhancer binding protein δ) has been implicated as a regulator of acute-phase response (APR) genes in hepatocytes. Its expression increases dramatically in liver during the APR and can be induced in hepatic cell lines by interleukin-6 (IL-6), an acute-phase mediator that activates transcription of many APR genes. Here we have investigated the mechanism by which C/EBPδ expression is regulated by IL-6 in hepatoma cells. C/EBPδ promoter sequences to− 125 bp are sufficient for IL-6 inducibility of a reporter gene and include an APR element (APRE) that is essential for IL-6 responsiveness. DNA binding experiments and transactivation assays demonstrate that Stat3, but not Stat1, interacts with this APRE. Two Sp1 sites, one of which is adjacent to the APRE, are required for IL-6 induction and transactivation by Stat3. Thus, Stat3 and Sp1 function cooperatively to activate the C/EBPδ promoter. Replacement of the APRE with Stat binding elements (SBEs) from the ICAM-1 or C/EBPβ promoter, both of which recognize both Stat1 and Stat3, confers responsiveness to gamma interferon, a cytokine that selectively activates Stat1. Sequence comparisons suggest that the distinct Stat binding specificities of the C/EBPδ and C/EBPβ SBEs are determined primarily by a single base pair difference. Our findings indicate that the cytokine specificity of C/EBPδ gene expression is governed by the APRE sequence.