MHC class II molecules present peptides, derived largely from exogenous antigens, to CD4+ T cells. Complex-generation occurs mainly in the specialized late endosomal MHC class II-rich compartment (MIIC) vesicles of antigen-presenting cells (APC). Dendritic cells (DC) have been reported to store intact antigen in MIIC until the receipt of an activation signal, when they process it into peptide-MHC class II complexes. However, constitutive migration of DC from the periphery to secondary lymphoid organs has been observed, and antigen presentation by nonactivated DC is proposed to play a role in the induction of tolerance to peripheral antigens. Thus, constitutive peptide-MHC class II complex generation must also occur in DC in immunologically quiescent situations. We have used a monoclonal antibody that detects a specific peptide-MHC class II complex to directly demonstrate constitutive complex generation in immature murine DC. Protein-derived peptide-MHC class II complexes were detected by flow cytometry at the DC surface within 1 h of antigen exposure in the absence of an exogenous activation signal, and could be detected by confocal microscopy in the MIIC within 5 min of antigen exposure. This processing activity was endotoxin independent. These data provide evidence for constitutive peptide-MHC class II complex generation in immature DC, and thus support a role for this activity in the induction of peripheral tolerance.