Cross-presentation of exogenous Ags in MHC class I molecules by dendritic cells is the underlying basis for many developing immunotherapies and vaccines. In the phagosome-to-cytosol pathway, Ags in phagocytosed particles must become freely soluble before being exported to the cytosol, but the kinetics of this process has yet to be fully appreciated. We demonstrate with a yeast vaccine model that the rate of Ag release in the phagosome directly affects cross-presentation efficiency, with an apparent time limit of approximately 25 min postphagocytosis for Ag release to be productive. Ag expressed on the yeast surface is cross-presented much more efficiently than Ag trapped in the yeast cytosol by the cell wall. The cross-presentation efficiency of yeast surface-displayed Ag can be increased by the insertion of linkers susceptible to cleavage in the early phagosome. Ags indirectly attached to yeast through Ab fragments are less efficiently cross-presented when the Ab dissociation rate is extremely slow.