NF-kappaB and STAT1 are critically involved in the initiation of the inflammatory cascade. Using semi-automated imaging cytometry and fluorescent antibodies, we screened several factors for their ability to induce nuclear translocation of RelA/NF-kappaB and STAT1 in subsets of monocyte-derived dendritic cells (DC). Detailed kinetics and dose-response studies are presented for IL-1-, LPS-, CD40L-, IFN-gamma- and IFN-alpha-stimulated responses. The results are consistent with the notion that simultaneous activation of both STAT1 and NF-kappaB pathways at the initiation of differentiation culture is required for efficient priming of IL-12 production by DC. Maturation of DC led to characteristic NF-kappaB and STAT1 distribution and response patterns. During the resting stage, DC differentiated under the presence of IFN-gamma showed sustained STAT activation and remained responsive to LPS. By contrast, PGE2-supplemented DC could be characterized by negligible responses to LPS and IFN-gamma and a remarkable NF-kappaB response to CD40L. STAT1 pathway was suppressed in PGE2-supplemented cells. We conclude that the magnitude and temporal kinetics of the nuclear shift of STAT1 and NF-kappaB in myeloid DC are associated with IL-12p70 production and are dependent on the nature of the stimulating factors and the polarization state of cells.