BID is an essential component of many apoptotic pathways. Cytosolic proteases cleave BID within an extended loop region, generating an active truncated fragment which synergizes with BAX and BAK to induce release of apoptogenic factors from mitochondria. To determine whether other proteins are cleaved in a similar manner as BID, we performed a database search for proteins which possess sequence similarity with the BID loop region. One of the proteins identified was the Hsc70-interacting protein (HIP). We analyzed the cleavage pattern of HIP using two known activators of BID: granzyme B and caspase-8. In in vitro cleavage assays using recombinant proteins, human and rat HIP were cleaved by granzyme B. Furthermore, the granzyme B-mediated cleavage site was mapped to the BID loop-like region of HIP by site-directed mutagenesis. This region was also the target for caspase-8-mediated cleavage in rat HIP. However, human HIP was not proteolyzed by caspase-8, which probably reflects sequence differences between human and rat HIP proteins at the P(1)' position of the caspase-8 recognition sequence. To determine whether HIP is cleaved during apoptosis, human Jurkat T cells were exposed to granzyme B and perforin. The results of these studies suggest that granzyme B-mediated loss of HIP expression occurs in vivo, and in a coordinate fashion with loss of BID, pro-caspase-8 and pro-caspase-3. These data implicate the Hsp70 co-chaperone HIP in the proteolytic cascade of some apoptotic pathways.