Cytotoxic elimination of dendritic cells (DC) in lymphoid tissue represents an important pathway of immune regulation. However, the mechanism of DC removal is still controversial since mature DC are insensitive to death receptor‐mediated killing and other surface or soluble molecules mediating DC death in vivo have yet to be characterized. Class II ligation is the only known signal that induces rapid cell death in mature DC, thus our studies have now focused on the requirements for this cell death using the advantages of tools available for both the mouse and human systems. Anti‐class II mAb could be grouped into (i) mAb that both bound to class II and caused class II‐mediated cell death as well as (ii) those that bound to class II, but did not cause apoptosis. mAb binding stable class II dimers as well as those mAb recognizing either the α or β chains of class II were found in both groups. Whereas class II‐mediated death was enhanced by DC–DC homotypic interactions, DC clustering itself was insufficient to induce apoptosis. Although DC death could be inhibited by uncoupling actin filament bundling, the inhibition of various proteases, including the caspases, and protein transport mediators failed to inhibit class II‐mediated cell death. Neither Bid, poly‐ADP‐ribose polymerase, caspases‐3, ‐7 and ‐8 nor FLICE‐inhibitory protein were found to be cleaved during class II apoptosis. Lastly, although class II mAb induced a rapid mitochondrial membrane depolarization in DC, cell death was not inhibited by Bcl‐2 over‐expression in DC. The independence of this form of apoptosis from protein or RNA synthesis, coupled to the rapidity of the mitochondrial depolarization and the lack of protection by Bcl‐2, suggests that mature DC express pre‐formed pro‐apoptotic molecules that are involved in class II‐mediated death.