A clinically relevant model of transient global brain ischemia involving cardiac arrest followed by resuscitation in dogs was utilized to study the expression and proteolytic processing of apoptosis-regulatory proteins. In the hippocampus, an increase in pro-apoptotic Bcl-2 family proteins Bcl-XS and Bak was detected, concomitant with proteolysis of Bcl-XL and Bcl-2, following ischemia-reperfusion injury. Also, biphasic cleavage of Bid was found in this region of the brain, with early generation of tBid-p11 within 10 min of cardiac arrest, followed by generation of tBid-p15 within 30-min reperfusion, consistent with activation of this pro-apoptotic protein. In addition, cardiac arrest and resuscitation induced early, reperfusion-dependent proteolytic processing of pro-caspase-6, -8, -10, and -14, which preceded caspase-3 activation. Immunohistochemical analysis using antibodies, which preferentially recognize processed caspase-3, -6, -8, and -10, provided evidence of time-dependent activation of these proteases in both neurons and glia in ischemia-sensitive regions of the brain. In conclusion, extremely rapid, cell-selective processing of apoptosis-regulatory proteins occurs in a clinically relevant model of ischemic brain injury caused by cardiac arrest and resuscitation. The early cleavage of Bid and rapid depletion of 32-kDa pro-caspase-14 from the canine hippocampus after induction of ischemia suggests the involvement of calpains in the processing of these proteins. Demonstration of in vitro cleavage of recombinant mouse caspase-14 by calpain I in the present study lends support to this hypothesis, further implicating cross-talk between different protease families in the pathophysiology of ischemic neural cell death.