LAP/NF-IL6 is a member of the C/EBP family of transcriptional activators and has been shown to be involved in the regulation of the acute-phase response. We have previously shown that phosphorylation of the liver-enriched transcriptional activator protein (LAP) Ser-105 enhances the activation of LAP-dependent genes. To identify the region which is important for gene activation, a series of LAP mutants were constructed, and domain swapping experiments with the DNA-binding domain of GAL4 were performed. These experiments point to an acidic region located between amino acids 21 and 105 of LAP/NF-IL6 which activates genes independent of the DNA-binding domain and the leucine zipper of LAP/NF-IL6. Computer-assisted predictions reveal two regions, a helical and a hydrophobic region in the transactivation domain, which could be important in mediating the direct interaction with the basal machinery. Site-directed mutagenesis of acidic residues in both regions demonstrates that the hydrophobic region located between amino acids 85 and 95 is the likely motif for the interaction with the basal machinery. Our results demonstrate that a hydrophobic region in the acidic transactivation domain of LAP/NF-IL6 seems to be relevant in mediating gene activation of LAP-dependent genes.