Interleukin 12 (IL-12) is a heterodimeric cytokine whose activity is critical for T-helper 1 responses. The gene for the IL-12 p40 subunit is expressed in macrophages following induction by bacterial products, and its expression is augmented by gamma interferon. In this study, we performed a functional analysis of the murine and human p40 promoters in the murine macrophage cell line RAW 264.7. Transcription from the murine p40 promoter was strongly induced by lipopolysaccharide and heat-killed Listeria monocytogenes (HKLM), but promoter activity was not enhanced by gamma interferon. Multiple cis-acting elements involved in activated transcription were identified through an extensive mutant analysis. The most critical element, whose activity is conserved in mice and humans, is located between positions -96 and -88 relative to the murine transcription start site. This element exhibits functional synergy with a previously described NF-kappaB half-site which interacts with Rel proteins. DNase I footprinting and electrophoretic mobility shift assays demonstrated that C/EBP proteins interact with the critical element, but in nuclear extracts, cooperative binding of C/EBP and Rel proteins to their respective sites was not observed. Interestingly, promoter activity was induced by HKLM in the presence of cycloheximide, consistent with induction by posttranslational mechanisms. The results suggest that C/EBP and Rel proteins play important roles in the activation of IL-12 p40 transcription by bacteria. However, many complex interactions will need to be clarified to fully understand p40 regulation.