The time-dependent changes of mitochondrial membrane potential and mass have been investigated on rat splenic lymphocytes stimulated with Con A in the presence and absence of reduced glutathione (GSH). Rhodamine-123 (Rh-123) and nonyl acridine orange (NAO) were used as specific dyes to monitor the membrane potential and mass of mitochondria, respectively. The percentage of cells showing blast transformation and the level of Rh-123 or NAO uptake were analyzed by flow cytometry. Present results demonstrate that a large number of cells showed activated mitochondria already at 24 hr after Con A stimulation and the activation of these organelles was not related to blast transformation. The addition of GSH into the culture medium increased the number of cells responding to mitogenic stimulation. In parallel it augmented the percentage of lymphocytes with activated mitochondria and also prevented their depolarization.