Signal-induced activation of caspases, the critical protease effectors of apoptosis, requires proteolytic processing of their inactive proenzymes. Consequently, regulation of procaspase processing is critical to apoptotic execution. We report here that baculovirus pancaspase inhibitor P35 and inhibitor of apoptosis Op-IAP prevent caspase activation in vivo, but at different steps. By monitoring proteolytic processing of endogenous Sfcaspase-1, an insect group II effector caspase, we show that Op-IAP blocked the first activation cleavage at TETD2G between the large and small caspase subunits. In contrast, P35 failed to affect this cleavage, but functioned downstream to block maturation cleavages (DXXD2(G/A)) of the large subunit. Substitution of P35's reactive site residues with TETDG failed to increase its effectiveness for blocking TETD2G processing of proSf-caspase-1, despite wild-type function for suppressing apoptosis. These data are consistent with the involvement of a novel initiator caspase that is resistant to P35, but directly or indirectly inhibitable by Op-IAP. The conservation of TETD2G processing sites among insect effector caspases, including Drosophila drICE and DCP-1, suggests that in vivo activation of these group II caspases involves a P35-insensitive caspase and supports a model wherein apical and effector caspases function through a proteolytic cascade to execute apoptosis in insects.The caspases are critical protease mediators of apoptosis and thus represent important targets for anti-apoptotic intervention (reviewed in Refs. 1-4). These highly conserved, aspartate-specific proteases are expressed as single-chain zymogens, which upon apoptotic signaling are activated by proteolytic processing, either by autoactivation, transactivation, or cleavage by other caspases (reviewed in Refs. 5 and 6). Once activated, the caspases proteolytically cleave a multitude of cellular substrates, leading to apoptotic death. Thus, caspase activation is a key regulatory point in the commitment to apoptosis.The molecular mechanisms regulating caspase activation are largely unknown. In mammals, a proteolytic cascade is initiated by group III caspases with long N-terminal prodomains (reviewed in Refs. 2, 3, 6, and 7). Upon activation, initiator caspases proteolytically cleave group II effector caspases at aspartate-containing sites through a regulated sequence of reactions that separate the large and small subunit, and then detach the short prodomain (reviewed in Ref. 4). To date, it is unknown whether analogous cascades are conserved in other organisms, including insects. Nevertheless, diverse apoptotic inhibitors from mammals, insects, and their associated viral pathogens are providing important insight into the regulatory mechanisms of caspase activation (8 -14).The baculoviruses encode two mechanistically distinct apoptotic suppressors, inhibitor of apoptosis (IAP) 1 and P35. Both viral proteins prevent premature insect cell death and thereby promote virus multiplication (reviewed in Ref. 15). The baculovirus...