Microbial molecules or cytokines can stimulate dendritic cell (DC) maturation, which involves DC migration to lymph nodes and enhanced presentation of Ag to launch T cell responses. Microbial TLR agonists are the most studied inducers of DC maturation, but type I IFN (IFN-I) also promotes DC maturation. In response to TLR stimulation, DC maturation involves a burst of Ag processing with enhanced expression of peptide-class II MHC complexes and costimulator molecules. Subsequently, class II MHC (MHC-II) synthesis and expression in intracellular vacuolar compartments is inhibited, decreasing Ag processing function. This limits presentation to a cohort of Ags kinetically associated with the maturation stimulus and excludes presentation of Ags subsequently experienced by the DC. In contrast, our studies show that IFN-I enhances DC expression of MHC-II and costimulatory molecules without a concomitant inhibition of subsequent MHC-II synthesis and Ag processing. Expression of mRNA for MHC-II and the transcription factor CIITA is inhibited in DCs treated with TLR agonists but maintained in cells treated with IFN-I. After stimulation with IFN-I, MHC-II expression is increased on the plasma membrane but is also maintained in intracellular vacuolar compartments, consistent with sustained Ag processing function. These findings suggest that IFN-I drives a distinctive DC maturation program that enhances Ag presentation to T cells without a shutdown of Ag processing, allowing continued sampling of Ags for presentation.