Exogenous proteins can be processed by antigen-presenting cells for the generation of MHC class I-restricted T cell responses. Where this occurs is not clear, although both transfer of internalized antigen into the cytosol and alternative processing in endolysosomes and phagosomes have been reported. Here we have studied the capacity of bone marrow-derived mouse myeloid dendritic cells (DC) to process the OVA protein for peptide presentation by H2-K(b). We have found that immature DC (iDC), both wild-type and transporter associated with antigen processing (TAP)-deficient cells, can transiently process OVA in a pathway which is resistant to inhibitors of the classical MHC class I pathway including the Golgi inhibitor Brefeldin A (BFA) and the proteasome inhibitor lactacystin. This alternative pathway is not found in subcultured DC with an intermediate maturity (imDC) or in resting, IL-3 expanded macrophages but can be re-expressed in imDC if these are activated by an immunostimulatory CpG oligonucleotide. Both iDC and CpG-activated DC were found to process OVA by regurgitation. In addition, we found that iDC secrete proteolytic enzymes into the supernatant, which can process OVA in the extracellular phase. These results suggest that multiple pathways exist for the processing of exogenous protein antigens into MHC class I-binding peptides.