Dendritic cells (DC), uniquely among APC, express an open/empty conformation of MHC class II (MHC-II) proteins (correctly folded molecules lacking bound peptides). Generation and trafficking of empty HLA-DR during DC differentiation are investigated here. HLA-DR did not fold as an empty molecule in the endoplasmic reticulum/trans-Golgi network, did not derived from MHC/Ii complexes trafficking to the cell surface, but was generated after invariant chain degradation within lysosomal-like MHC-II rich compartments (MIIC). In pre-DC, generated from monocytes cultured in the presence of GM-CSF, Lamp-1(+)MHC-II(+) compartments are predominantly electron dense and, in these cells, empty MHC-II molecules accounts for as much as 20% of total surface HLA-DR. In immature DC, generated in presence of GM-CSF and IL-4, empty HLA-DR reside in multilamellar MIIC, but are scarcely observed at the cell surface. Thus, the morphology/composition of lysosomal MIIC at different DC maturational stages appear important for surface egression or intracellular retention of empty HLA-DR. Ag loading can be achieved for the fraction of empty HLA-DR present in the "peptide-receptive" form. Finally, in vivo, APC-expressing surface empty HLA-DR were found in T cell areas of secondary lymphoid organs.