An emerging area of research has demonstrated that plasminogen functions in the acute-phase response to tissue injury, neoplastic growth or infection. We have previously shown that the acute-phase mediator, interleukin (IL)-6, increases circulating plasminogen levels via upregulation of plasminogen promoter activity. We also identified a putative IL-6 responsive element (nt -791 to -783; IL6-RE) in the plasminogen gene that is required for maximal stimulation of promoter activity by IL-6. For the present study, we investigated the transcription factors and signaling pathway mediating the response of the plasminogen gene to IL-6. In electrophoretic mobility shift assays (EMSAs), a radiolabeled oligonucleotide IL6-RE probe formed specific complexes with nuclear proteins from untreated hepatocytic cells. The extent of complex formation was markedly increased using nuclear proteins from IL-6-treated cells. Complex formation was abolished by an oligonucleotide with the consensus CCAAT/enhancer binding protein (C/EBP) sequence. Furthermore, complexes were supershifted by antibodies to C/EBPbeta. Treatment of Hepa 1-6 cells with the mitogen-activated protein kinase (MAPK) inhibitor, PD-98059, inhibited IL-6-stimulated plasminogen promoter activity. These results suggest that transcription factor C/EBPbeta and the MAPK pathway play key roles in the response of the plasminogen gene to IL-6, thus elucidating a major mechanism by which the plasminogen system is upregulated to perform its crucial functions in the acute-phase response.