Tumor necrosis factor alpha (TNFα) is a key regulatory cytokine whose expression is controlled by a complex set of stimuli in a variety of cell types. Previously, we found that the monocyte/macrophage-enriched nuclear transcription factor C/EBPβ played an important role in the regulation of the TNFα gene in myelomonocytic cells. Abundant evidence suggests that other transcription factors participate as well. Here we have analyzed interactions between C/EBPβ and c-Jun, a component of the ubiquitously expressed AP-1 complex. In phorbol myristate acetate (PMA)-treated Jurkat T cells, which did not possess endogenous C/EBPβ, expression of c-Jun by itself had relatively little effect on TNFα promoter activity. However, the combination of C/EBPβ and c-Jun was synergistic, resulting in greater than 130-fold activation. This effect required both the leucine zipper and DNA binding domains, but not the transactivation domain, of c-Jun, plus the AP-1 binding site centered 102/103 bp upstream of the transcription start site in the TNFα promoter. To determine if C/EBPβ and c-Jun might cooperate to regulate the cellular TNFα gene in myelomonocytic cells, U937 cells that possess endogenous C/EBPβ and were stably transfected with either wild-type c-Jun or the transactivation domain deletion mutant (TAM-67) were examined. U937 cells expressing ectopic wild-type c-Jun or TAM-67 secreted over threefold more TNFα than the control line in response to PMA plus lipopolysaccharide. Transient transfection of the U937 cells expressing TAM-67 suggested that TAM-67 binding to the -106/- 99-bp AP-1 binding site cooperated with endogenous C/EBPβ in the activation of the -120 TNFα promoter-reporter. DNA binding assays using oligonucleotides derived from the TNFα promoter suggested that C/EBPβ and c-Jun interact in vitro and that the interaction may be DNA dependent. Our data demonstrate that the TNFα gene is regulated by the interaction of the ubiquitous AP-1 complex protein c-Jun and the monocyte/macrophage-enriched transcription factor C/EBPβ and that this interaction contributes to the expression of the cellular TNFα gene in myelomonocytic cells. This interaction was unique in that it did not require the c-Jun transactivation domain, providing new insight into the cell-type-specific regulation of the TNFα gene.