The P35 protein derived from the baculovirus Autographa californica NPV has been characterized as an inhibitor of apoptotic cell death in a great number of organisms and situations. This potential has been further mapped to the capacity of P35 to inhibit all caspases investigated. Here we show that P35 does not inhibit caspase-9 activity in a cell-free system of mammalian caspase activation. In cell extracts, cytochrome c addition led to the activation of caspase-9, -3 and -7. When cytosolic extract from cells expressing P35 was added, caspase-9-mediated maturation of caspase-3 proceeded normally but caspase-3-mediated further events were prevented, such as complete processing of caspase-3, processing of caspase-7 and the appearance of DEVD-cleaving activity. The P35 protein from Bombyx mori NPV, which has been reported to have a much weaker anti-apoptosis activity in vivo was found also to have significant caspase-3-inhibiting activity. These data suggest that P35 evolved specifically to inhibit effector rather than initiator caspases.