The mechanism of the induction of apoptosis by arsenic trioxide (As2O3), which was demonstrated recently to be an effective inducer of apoptosis in patients with leukemia, was examined in detail in human leukemia U937 cells. Upon treatment of U937 cells with 50 microM of As2O3, complete inactivation of the kinases ERK1 and ERK2 was detected within 30 min. p38 was activated within 3 hr, and the maximum activity was detected at 6 hr, when DNA fragmentation remained undetectable. Experiments with transfected cells that expressed constitutively activated MEK1 and a specific inhibitor of p38 also suggested that inactivation of ERKs and activation of p38 might be associated with the induction of apoptosis by As2O3. In contrast to the inactivation of ERKs and the activation of p38, activation of JNK by As2O3 appeared to protect cells against the induction of apoptosis. Treatment of U937 cells with As2O3 also caused the Ca2+-dependent production of superoxide and intracellular acidification and a decrease in the mitochondrial membrane potential at the early stages of induction of apoptosis by As2O3. These changes preceded the release of cytochrome c from mitochondria and the activation of caspase-3. It should be possible to exploit the unusual characteristics of the mechanism of induction of apoptosis by As2O3 in U937 cells by making use of synergistic effects of this compound with other inducers of apoptosis.