The present study demonstrates that monocyte-derived dendritic cells (moDCs) produced in vitro using a GM-CSF and IFN-α differentiation protocol encompass a rare (T}5%) subpopulation of cells showing classical dendritic cell morphology and capable of natural internalization of extracellular self-DNA.We established that DEFB, HMGB1, LL-37 and RAGE antigens, which mediate the process of DNA internalization, are expressed on the surface of moDCs similar to plasmacytoid dendritic cells. However, in constrast to the latter subpopulation, these cells do not produce interleukin (IL)-37. Nonetheless, the process of DNA internalization was not in direct relation to the presence of the above antigens on the surface of these cells. Dendritic cells were sorted into total and non-DNA-internalizing populations and cytokine production was analyzed at 24-48 hours post-DNA treatment. We show that massive secretion of cytokines by dendritic cells is associated with the dsDNA-internalizing subpopulation. A total pool of IFNmoDCs secrete pro-inflammatory “first-wave” cytokines (IL-2, IL-6, IL-8, TNF-α) at both 24 and 48 hours time points. The anti-inflammatory cytokines IL-4 and IL-10 were found to be modestly induced, whereas GM-CSF, GCSF, and IFN-γ production was strongly induced. Treatment of moDCs with dsDNA results in the up-regulated transcription of IFN-α, IFN-β, IFN-γ, IL-8, IL-10, and VEGF by 6 hours. Combined dsDNA + chloroquine treatment has a synergistic effect on transcription of only one of the genes tested, with the pro-inflammatory cytokine IFN-b displaying the strongest fold induction by 24 hours.