CD8+ T cells are a key immune component for the eradication of many intracellular pathogens. This study aims to characterize the human CD8 + T cell response to naturally processed chlamydial Ags in individuals exposed to the intracellular pathogen Chlamydia trachomatis. By using C. trachomatis-infected autologous dendritic cells (DCs) as stimulators, Chlamydia-reactive CD8+ T cell responses were detected in all 10 individuals tested. The majority of the Chlamydia-reactive CD8+ T cells were non-MHC class Ia restricted in all three of the individuals tested. From one donor, three non-class Ia-restricted and two class Ia-restricted Chlamydia-specific CD8+ T cells were cloned and characterized further. All five T cell clones secreted IFN-γ in response to autologous DCs infected with viable Chlamydia, but not with DCs pulsed with inactivated chlamydial elementary bodies. MHC class Ia-restricted and non-class Ia-restricted responses were inhibited by DC treatment with a proteasomal inhibitor and an endoplasmic reticulum-Golgi transport inhibitor, suggesting that these T cells recognize a peptide Ag translocated to the host cell cytosol during infection that is processed via the classical class Ia Ag-processing pathway. Even though both restricted and nonrestricted CD8+ T cells produced IFN-γ in response to Chlamydia-infected fibroblasts, only the non-class Ia-restricted cells were lytic for these targets. The class Ia-restricted CTLs, however, were capable of cytolysis as measured by redirected killing. Collectively, these data demonstrate that both class Ia-restricted and non-classically restricted CD8+ T cells are elicited in C. trachomatis-exposed individuals. Their role in host immunity remains to be elucidated.