Vaccination against cancer or intracellular pathogens requires stimulation of class I-restricted CD8(+) T cells. It is therefore important to develop Ag delivery vectors that will promote cross-presentation by APCs and stimulate appropriate inflammatory responses. Toward this goal, we tested the potential of Escherichia coli as an Ag delivery vector in in vitro human culture. Bacteria expressing enhanced green fluorescent protein were internalized efficiently by dendritic cells, as shown by flow cytometry and fluorescence microscopy. Phenotypic changes in DC were observed, including up-regulation of costimulatory molecules and IL-12p40 production. We tested whether bacteria expressing recombinant Ags could stimulate human T cells using the influenza matrix protein as a model Ag. Specific responses against an immunodominant epitope were seen using IFN-gamma ELISPOT assays when the matrix protein was coexpressed with listeriolysin O, but not when expressed alone. THP-1 macrophages were also capable of stimulating T cells after uptake of bacteria, but showed slower kinetics and lower overall levels of T cell stimulation than dendritic cells. Increased phagocytosis of bacteria induced by differentiation of THP-1 increased their ability to stimulate T cells, as did opsonization. Presentation was blocked by proteasome inhibitors, but not by lysosomal protease inhibitors leupeptin and E64. These results demonstrate that recombinant E. coli can be engineered to direct Ags to the cytosol of human phagocytic APCs, and suggest possible vaccine strategies for generating CD8(+) T cell responses against pathogens or tumors.