Interleukin-12 (IL-12) and IL-23 are heterodimeric cytokines that serve as critical regulators of T helper cell development. The Il12b gene, which encodes the p40 subunit of both IL-12 and IL-23, is expressed in macrophages and dendritic cells following induction by bacterial products. Although the Il12b promoter, like the promoters of most proinflammatory genes, can support transcriptional induction in typical transfection assays, we show that it is not sufficient for transcription in an insulated chromatin environment. Using a DNase I hypersensitivity assay, two potential distal control regions were identified. One region, DNase I-hypersensitive site 1 (HSS1), located 10 kb upstream of the transcription start site, exhibited hypersensitivity only in stimulated macrophages. In an insulated environment, a 105-bp fragment spanning HSS1 was sufficient for transcription when combined with the Il12b promoter. Although several elements are likely to contribute to activity of the endogenous HSS1 enhancer, including an evolutionarily conserved binding site for C/EBP proteins, the only element required for activity in transient- and stable-transfection assays bound Oct-1 and Oct-2, both of which are expressed constitutively in macrophages. Oct-1 and Oct-2 were recruited to the enhancer upon macrophage stimulation, and the Oct site appeared important for nucleosome remodeling at HSS1. These results suggest that the HSS1 enhancer and Oct proteins play central roles in Il12b induction upon macrophage activation.