We have recently shown that lymphocyte apoptosis induced by dexamethasone or superantigens is accompanied by reduction of mitochondrial transmembrane potential (ΔΨ_m) which precedes nuclear DNA fragmentation. Here, we demonstrate that fluorochromes such as 3,3′dihexyloxacarbocyanine iodide [DiOC₆(3)] which measure ΔΨ_m, or fluorochromes such as hydroethidine (HE) which measure mitochondrial superoxide anion production allow the identification of thymocytes or peripheral T lymphocytes which are eliminated by apoptosis in vivo. In mice bearing transgenic α/β T cell receptor (TCR) specific for a class I‐restricted male‐specific peptide, the superoxide‐mediated oxidation of HE into ethidium (Eth) is enhanced among thymocytes which are being deleted due to negative selection (CD4⁺ CD8⁺ cells expressing the transgenic TCR in male mice) or lack of positive selection (CD4⁺ CD8⁻ thymocytes from female mice). ΔΨ_m reduction and/or enhanced HE oxidation are also found when apoptosis is induced by a series of pathogenic agents. Thus, lethal irradiation provokes mitochondrial and nuclear signs of apoptosis, and both these alterations are absent in mice bearing a p53 null mutation, underlying the correlation between mitochondrial perturbation and nuclear apoptosis. Similarly, superantigen‐triggered deletion of peripheral T cells in vivo is accompanied by enhanced HE → Eth conversion and reduced DiOC₆(3) uptake. More importantly, as compared to normal controls. CD4⁺ or CD8⁺ cells from clinically asymptomatic human immunodeficiency virus‐1 (HIV‐1) carriers also contain a significantly elevated percentage of cells endowed with reduced DiOC₆(3) uptake. In superantigen‐ and HIV‐induced apoptosis, the percentage of T lymphocytes with a subnormal DiOC₆(3) uptake is more important than that of cells marked by enhanced HE → Eth conversion. In conclusion, mitochondrial alterations precede and/or accompany nuclear signs of apoptosis induced by physiological and a variety of different pathogenic agents in vivo.