(Background and aim) Interleukin (IL)-32 is a recently described proinflammatory cytokine, characterized by the induction of nuclear factor (NF)-B activation. We studied IL-32 expression in human pancreatic periacinar myofibroblasts, which play important roles in the regulation of extracellular matrix metabolism and inflammatory responses in the pancreas. (Methods) IL-32 protein expression was evaluated by Western blot analyses, and IL-32 mRNA expression was analyzed by Northern blot and real-time PCR analyses. (Results) IL-32 mRNA was weakly expressed without a stimulus, and its expression was markedly enhanced by IL-1, IFN- and TNF-. IL-1, IFN- and TNF- enhanced intracellular accumulation of IL-32 protein, but IL-32 was not detected in supernatants. Each cytokine dose- and time-dependently induced IL-32 mRNA expression. An inhibitor of phosphatidylinositol 3-kinase (LY294002) significantly suppressed IL-1-, IFN-- and TNF--induced IL-32 mRNA expression, although MAPK inhibitors had no effect. Akt activation in response to these cytokines was confirmed by Western blot. Furthermore, LY294002 suppressed both IL-1- and TNF--induced NF-B activation and IL-1-, TNF-- and IFN--induced AP-1 activation. Blockade of NF-B and AP-1 activation by an adenovirus expressing a stable mutant form of IB and a dominant negative mutant of c-Jun markedly suppressed IL-1-, IFN-- and/or TNF--induced IL-32 mRNA expression. (Conclusions) Human pancreatic periacinar myofibroblasts expressed IL-32 in response to IL-1, TNF- and IFN-. IL-32 mRNA expression is dependent on interactions between the PI3K/Akt-pathway and the NF-B/AP-1 system.