Pathogenic mycobacteria circumvent host immune responses by disruption of macrophage effector functions. Here we examined cytokine production, gene expression and protein-DNA interactions of MAP-infected murine J774A.1 and RAW264.7 macrophage cell lines (MAPMac) and compared it to macrophages stimulated with LPS (LPSMac), a potent activator of macrophages. ELISA and Northern analyses revealed that the expression levels of TNFα, IL-1β, and IL-6 were significantly lower in MAPMac than in LPSMac. IL-6 was almost not induced in MAPMac RAW264.7 cells, whereas its expression increased over the time in MAPMac J774A.1 cells. Since the transcription regulators NFκB and C/EBPβ contribute to IL-6 gene expression electrophoretic-mobility-shift-assays (EMSA) were performed. EMSA revealed reduced NFκB and C/EBPβ binding to the NFκB and C/EBP binding site of the IL-6 gene in MAPMac as compared to LPSMac. Interestingly, when compared to uninfected cells C/EBPβ binding activity was almost not enhanced in MAPMac RAW264.7 cells but enhanced in MAPMac J774.A1 cells. In these cells, however, C/EBPβ binding complexes appeared to be smaller than in LPSMac. C/EBPβ protein complexes can be composed of two alternatively translated C/EBPβ-isoforms; the larger 37 kDa LAP that is fully transactive and the smaller 20 kDa LIP that has reduced transactivating capacity. Thus, different LAP-LIP combinations will result in C/EBPβ EMSA complexes of different sizes and biological function. Since C/EBPβ mRNA expression was similar in LPSMac and MAPMac, we determined the LAP-LIP ratio in nuclear extracts from LPSMac and MAPMac by Southwestern analysis. We found LAP-LIP ratios similar to that of untreated cells in MAPMac, whereas the ratios were considerably higher in LPSMac. These data provide evidence for a reduced transactivating capacity of C/EBPβ leading to reduced IL-6 expression in MAPMac.