Yeasts of the genus Saccharomyces expressing recombinant antigens are currently evaluated as candidate T cell vaccines. Here, we compared the interaction kinetics between four biotechnologically relevant yeast genera (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis and Pichia pastoris) and human dendritic cells as well as the involvement of Dectin-1 and mannose receptor in phagocytosis. Further, we analyzed the activation capacity of recombinant yeasts expressing ovalbumin (OVA) either intracellular, extracellular or surface-displayed by OVA-specific CD8 T lymphocytes. We found that the kinetic patterns of yeast uptake by phagocytic cells varied between the tested yeast genera and that both genus and subcellular OVA antigen localization influenced the strength of T cell activation. In particular, in S. cerevisiae, a secreted antigen was less effectively delivered than its cytosolic variant, whereas most efficient antigen delivery with P. pastoris was obtained by cell surface bound antigen. Our data indicate that protein secretion might not be an effective delivery pathway in yeast.