INTRODUCTION: Immune system dysregulation has been demonstrated to occur during and immediately following spaceflight. If found to persist during lengthy flights, this phenomenon could be a serious health risk to crewmembers participating in lunar or Mars missions. METHODS: A comprehensive postflight immune assessment was performed on 17 short-duration Space Shuttle crewmembers and 8 long-duration International Space Station (ISS) crewmembers. Testing consisted of peripheral leukocyte subset analysis, early T cell activation potential, and intracellular/secreted cytokine profiles. RESULTS: For Shuttle crewmembers, the distribution of the peripheral leukocyte subsets was found to be altered postflight. Early T cell activation was elevated postflight; however, the percentage of T cell subsets capable of being stimulated to produce IL-2 and IFN gamma was decreased. The ratio of secreted IFN gamma:IL-10 following T cell stimulation declined after landing, indicating a Th2 shift. For the ISS crewmembers, some alterations in peripheral leukocyte distribution were also detected after landing. In contrast to Shuttle crewmembers, the ISS crewmembers demonstrated a statistically significant reduction in early T cell activation potential immediately postflight. The percentage of T cells capable of producing IL-2 was reduced, but IFN gamma percentages were unchanged. A reduction in the secreted IFN gamma:IL-10 ratio (Th2 shift) was also observed postflight in the ISS crewmembers. CONCLUSION: These data indicate that consistent peripheral phenotype changes and altered cytokine production profiles occur following spaceflight of both short and long duration; however, functional immune dysregulation may vary related to mission duration. In addition, a detectable Th2 cytokine shift appears to be associated with spaceflight.