Bz-423 is a 1,4-benzodiazepine with selective lymphotoxic properties and potent therapeutic activity against lupus-like disease in autoimmune mice. In NZB/W lupus-prone mice, Bz-423 specifically kills germinal center B cells, which are the cells that drive disease both in this model and in human systemic lupus erythematosus. In this report, the mechanistic basis for the selective action of Bz-423 is investigated. We show that Bz-423-induces superoxide as an immediate early response and that this reactive oxygen species is more effective as a second messenger death signal in B cells activated by B cell receptor stimulation compared with resting cells. As a result, low [Bz-423] that are not cytotoxic to non-stimulated cells kill stimulated cells in synergy with anti-immunoglobulin M antibodies. Subsequent experiments demonstrated that Bz-423 extends the rise in intracellular calcium that accompanies anti-immunoglobulin M stimulation, and this effect mediates the synergistic death response. Because B cell hyperactivation and altered calcium signaling is a distinguishing feature of autoreactive lymphocytes in lupus, the mechanism by which Bz-423 induces apoptosis preferentially targets disease-causing cells on the basis of their activation state. Thus, molecules like Bz-423 could form the basis for new and selective anti-lupus agents.