The anti-apoptotic protein p35 from baculovirus is thought to prevent the suicidal response of infected insect cells by inhibiting caspases. Ectopic expression of p35 in a number of transgenic animals or cell lines is also anti-apoptotic, giving rise to the hypothesis that the protein is a general inhibitor of caspases. We have verified this hypothesis by demonstrating that purified recombinant p35 inhibits human caspase-1, -3, -6, -7, -8, and -10 with kass values from 1.2 x 10(3) to 7 x 10(5) (M-1 s-1), and with upper limits of Ki values from 0.1 to 9 nM. Inhibition of 12 unrelated serine or cysteine proteases was insignificant, implying that p35 is a potent caspase-specific inhibitor. Mutation of the putative inhibitory loop to favor caspase-1 resulted in a substantial decline in caspase-3 inhibition, but minimal changes in caspase-1 inhibition. The interaction p35 with caspase-3, as a model of the inhibitory mechanism, revealed classic slow-binding inhibition, with both active-sites of the caspase-3 dimer acting equally and independently. Inhibition resulted from complex formation between the enzyme and inhibitor, which could be visualized under nondenaturing conditions, but was dissociated by SDS to give p35 cleaved at Asp87, the P1 residue of the inhibitor. Complex formation requires the substrate-binding cleft to be unoccupied. Taken together, these data revealed that p35 is an active-site-directed inhibitor highly adapted to inhibiting caspases.