CD8+ T cells use a number of effector mechanisms to protect the host against infection. We have studied human CD8+ T cells specific for CMV pp65 495–503 epitope, or for staphylococcal enterotoxin B, for the expression patterns of five cytokines and cytolytic effector molecules before and after antigenic stimulation. Ex vivo, the cytolytic molecule granzyme B was detected in a majority of circulating CMV-specific CD8+ T cells, whereas perforin was rarely expressed. Both were highly expressed after Ag-specific activation accompanied by CD45RO up-regulation. TNF-α, IFN γ, and IL-2 were sequentially acquired on recognition of Ag, but surprisingly, only around half of the CMV-specific CD8+ T cells responded to antigenic stimuli with production of any cytokine measured. A dominant population coexpressed TNF-α and IFN-γ, and cells expressing TNF-α only, IFN-γ only, or all three cytokines together also occurred at lower but clearly detectable frequencies. Interestingly, perforin expression and production of IFN-γ and TNF-α in CD8+ T cells responding to staphylococcal enterotoxin B appeared to be largely segregated, and no IL-2 was detected in perforin-positive cells. Together, these data indicate that human CD8+ T cells can be functionally segregated in vivo and have implications for the understanding of human CD8+ T cell differentiation and specialization and regulation of effector mechanisms.