Cancer vaccines aim to induce antitumor CTL responses, which require cross-presentation of tumor Ag to CTLs by dendritic cells (DCs). Adjuvants that facilitate cross-presentation of vaccine Ag are therefore key for inducing antitumor immunity. We previously reported that human DCs could not efficiently cross-present the full-length cancer/testis Ag NY-ESO-1 to CTL unless formulated as either an immune complex (NY-ESO-1/IC) or with ISCOMATRIX adjuvant. We now demonstrate that NY-ESO-1/ICs induce cross-presentation of HLA-A2- and HLA-Cw3-restricted epitopes via a proteasome-dependent pathway. In contrast, cross-presentation of NY-ESO-1/ISCOMATRIX vaccine was proteasome independent and required the cytosolic protease tripeptidyl peptidase II. Trafficking studies revealed that uptake of ICs and ISCOMATRIX vaccine by DCs occurred via endocytosis with delivery to lysosomes. Interestingly, ICs were retained in lysosomes, whereas ISCOMATRIX adjuvant induced rapid Ag translocation into the cytosol. Ag translocation was dependent on endosomal acidification and IL-4-driven differentiation of monocytes into DCs. This study demonstrates that Ag formulation determines Ag processing and supports a role for tripeptidyl peptidase II in cross-presentation of CTL epitopes restricted to diverse HLA alleles.