Phenylarsine oxide (PAO), a membrane-permeable trivalent arsenical, is widely used as an inhibitor of protein tyrosine phosphatases. It reacts with vicinal sulfhydryl groups of proteins to form stable ring structures. Here we show the regulatory function of PAO in immune responses from macrophages. PAO significantly induced the secretion of interleukin (IL)-12p40 in lipopolysaccharide-stimulated macrophages. The mRNA expression and the gene promoter activity of IL-12p40 were enhanced by PAO. These results suggest that PAO may enhance IL-12p40 production at the transcriptional level. Furthermore, the effects of PAO on several signaling molecules regulating IL-12p40 expression were investigated. PAO attenuated the induced binding activity of cAMP response element (CRE), but not of NF-kB. Moreover, CRE promoter activity was dose-dependently inhibited by PAO and the increased secretion of IL-12p40 by PAO was reduced by forskolin, a cAMP activator. These results suggest that PAO enhances IL-12p40 production by inhibiting CRE activity.