Monocyte-derived dendritic cells (DCs) are used as adjuvant cells in cancer immunotherapy and have shown promising results. In order to obtain full functional capacity, these DCs need to be maturated, and the current “gold standard” for this process is maturation with TNF-α, IL-1β, IL-6 and PGE 2 used for generating standard DCs (sDC). Several studies indicate that IFN-α might also be important for DC differentiation and maturation. In this study, we tested the effect of IFN-α alone or as addition to the gold standard sDC cocktail. We observed that maturation by IFN-α differs from sDC maturation: The major phenotypic change after IFN-α maturation was dose-dependent up-regulation of CD38 but not CD83, while sDCs expressed the opposite profile with low CD38 and high CD83 expression. Similarly, maturation by Poly I:C leads to CD38high, CD83low DCs indicating a functional relationship between CD38, IFN-α and TLR3. Thus, CD38 appear to be a relevant marker for activation by TLR3 or IFN-α. Addition of IFN-α to the sDC cocktail results in up-regulation of both CD38 and CD83 and improved capacity for induction of autologous T-cell responses despite few other changes in DC phenotype and cytokine secretion. Our observations suggest that IFN-α could be included in maturation protocols for clinical grade DCs used for immunotherapy against cancer and should be included if DCs are used for CD8+ T-cell stimulation in vitro.