The ability of virus-specific CD8(+) T cells to produce cytokines was studied in mice infected with lymphocytic choriomeningitis virus and vesicular stomatitis virus. Intracellular staining was used to visualize cytokine-producing CD8(+) and CD4(+) T cells. Overall, virus-specific CD8(+) T cells produce a similar range of cytokines (IFN-gamma, TNF-alpha, IL-2, GM-CSF, RANTES, MIP-1alpha and MIP-1beta) as CD4(+) T cells, but the relative distribution of cytokine-producing subsets is different. Moreover, cytokine-producing CD8(+) T cells were found to dominate numerically at all time-points tested. Co-staining for more than one cytokine revealed that while all cytokine-producing CD8(+) T cells synthesized IFN-gamma, additional cytokines were produced by partly overlapping subsets of this population. The frequency of cells producing more than one cytokine was higher in a tertiary site (peritoneum) and generally increased with transition into the memory phase; however, GM-CSF producing cells were only present transiently. Concerning factors predicted to influence the distribution of cytokine-producing subsets, IFN-gamma and IL-12 did not play a role, nor was extensive virus replication essential. Notably, regarding the heterogeneity in cytokine production by individual cells with similar epitope specificity, variation in TCR avidity was not the cause, since in vivo-activated TCR transgene-expressing cells were as heterogeneous in cytokine expression as polyclonal cells specific for the same epitope.