The immune system is highly sensitive to stressors present during spaceflight. The major emphasis of this study was on the T lymphocytes in C57BL/6NTac mice after return from a 13-day space shuttle mission (STS-118). Spleens and thymuses from flight animals (FLT) and ground controls similarly housed in animal enclosure modules (AEM) were evaluated within 3-6 h after landing. Phytohemagglutinin-induced splenocyte DNA synthesis was significantly reduced in FLT mice when based on both counts per minute and stimulation indices (P < 0.05). Flow cytometry showed that CD3⁺ T and CD19⁺ B cell counts were low in spleens from the FLT group, whereas the number of NK1.1⁺ natural killer (NK) cells was increased (P < 0.01 for all three populations vs. AEM). The numerical changes resulted in a low percentage of T cells and high percentage of NK cells in FLT animals (P < 0.05). After activation of spleen cells with anti-CD3 monoclonal antibody, interleukin-2 (IL-2) was decreased, but IL-10, interferon-, and macrophage inflammatory protein-1 were increased in FLT mice (P < 0.05). Analysis of cancer-related genes in the thymus showed that the expression of 30/84 genes was significantly affected by flight (P < 0.05). Genes that differed from AEM controls by at least 1.5-fold were: Birc5, Figf, Grb2, and Tert (up-regulated) and Fos, Ifnb1, Itgb3, Mmp9, Myc, Pdgfb, S100a4, Thbs, and Tnf (down-regulated). Collectively, the data show that T cell distribution, function, and gene expression are significantly modified shortly after return from the spaceflight environment.