Human immunodeficiency virus (HIV)-related opportunistic infections continue to occur in patients who are newly diagnosed with HIV infection, those in the early course of highly active antiretroviral therapy or nonadherent to HIV care, and other immunosuppressed individuals. One of the most common opportunistic infections in these patients is Pneumocystis pneumonia. CD8⁺ T cells are recruited to the lung after P. carinii infection and have been associated with both lung injury and host defense. This variability may be due to subpopulations of CD8⁺ T cells recruited to the lung. We have previously shown using adoptive transfer studies that in vivo-generated T-cytotoxic-1 (Tc1) CD8⁺ T cells, defined by the secretion of gamma interferon (IFN-γ), have effector activity against Pneumocystis spp. in vitro as well as in vivo. To better understand the mechanisms of these effects, we generated, expanded, and tested Tc1 and Tc2 CD8⁺ T cells specific for P. murina ex vivo. Tc1-polarized CD8⁺ T cells secreted higher levels of IFN-γ and granulocyte-macrophage colony-stimulating factor (GM-CSF) and lower levels of interleukin-4 (IL-4), IL-5, IL-10, and IL-13 than Tc2 CD8⁺ T cells when stimulated with P. murina antigen. Moreover, Tc1 CD8⁺ T cells demonstrated enhanced effector activity in a macrophage-mediated killing assay which was independent of cell contact. The augmentation in macrophage-mediated P. murina killing was significantly abrogated when GM-CSF was neutralized in the Tc1 CD8⁺ T cells. These data support the possibility that antigen-specific GM-CSF secretion is critical for effector activity of P. murina-specific Tc1 CD8⁺ T cells in vitro.