Although studies with primary lymphocytes are almost always conducted in CO₂ incubators maintained at atmospheric oxygen levels (atmosO₂; 20%), the physiological oxygen levels (physO₂; 5%) that cells encounter in vivo are 2–4 times lower. We show here that culturing primary T cells at atmosO₂ significantly alters the intracellular redox state (decreases intracellular glutathione, increases oxidized intracellular glutathione), whereas culturing at physO₂ maintains the intracellular redox environment (intracellular glutathione/oxidized intracellular glutathione) close to its in vivo status. Furthermore, we show that CD3/CD28-induced T cell proliferation (based on proliferation index and cell yield) is higher at atmosO₂ than at physO₂. This apparently paradoxical finding, we suggest, may be explained by two additional findings with CD3/CD28-stimulated T cells: (i) the intracellular NO (iNO) levels are higher at physO₂ than at atmosO₂; and (ii) the peak expression of CD69 is significantly delayed and more sustained at physO₂ that at atmosO₂. Because high levels of intracellular NO and sustained CD69 tend to down-regulate T cell responses in vivo, the lower proliferative T cell responses at physO₂ likely reflect the in vitro operation of the natural in vivo regulatory mechanisms. Thus, we suggest caution in culturing primary lymphocytes at atmosO₂ because the requisite adaptation to nonphysiological oxygen levels may seriously skew T cell responses, particularly after several days in culture.