Interleukin-23 (IL-23) is a heterodimeric cytokine composed of the p40 and p19 subunits, the first of which is also part of the IL-12 heterodimer. IL-23 induces a unique T helper cell subset to produce IL-17, which plays a critical and IL-12/IFN-gamma-independent role in autoimmunity. Plasmacytoid dendritic cells (pDC), as opposed to myeloid DC (mDC) and the closely related epidermal Langerhans cells (LC), exhibit a specific and broad range of pro-inflammatory cytokine secretion, with type I interferons representing a typical difference to classical mDC and LC. In this study we show that upon treatment with a selection of ligands for Toll-like receptor (TLR) 3, 4, 7, and 9, only mDC and LC but not pDC secreted IL-23. While pDC produced both mRNA and protein of the p40 subunit, the lack of bioactive heterodimeric IL-23 protein release was related to the fact that in these cells only the p19 mRNA was expressed which was not translated into protein. In addition to these differential findings in both DC subsets a novel p19 splice variant was identified. This analysis of transcriptional and/or post-transcriptional regulation of the IL-23 subunits p40 and p19 may help to understand the complex regulation of heterodimeric cytokines and the overlapping but distinct functions of IL-12 and IL-23. It supports the hypothesis of a coordinated adaptive immune response based on a finely tuned contribution of these cytokines by different mouse DC subsets.