Although caspase-8 has an established role as an initiator of death receptor-mediated apoptosis, the function of its closest homolog, caspase-10, is almost completely unknown. To gain a closer insight into the physiological function of caspase-10, we compared the cleavage of known caspase-8 substrates by both initiator caspases. We demonstrate that caspase-10 and -8 have overlapping cleavage preferences for several substrates such as the kinases RIP and PAK2. Interestingly, in other substrates, such as the Bcl-2 protein Bid, we found additional and distinct cleavage sites for both caspases, which might have important consequences for mitochondrial targeting and propagation of the death signal. Caspase-8 and -10 also caused different interchain cleavage patterns of their enzyme precursors. Together, these results suggest that caspase-8 and -10, despite having overlapping functions, also have selective substrate cleavage specificities and might thereby exert nonredundant roles in apoptosis signaling.