The p35 protein from baculovirus is a potent, broad-spectrum inhibitor of caspases that blocks host cell death prior to viral replication. Like a Serpin, p35 presents a reactive site loop that undergoes a large conformational change upon cleavage by caspases, resulting in an SDS-stable complex. Structural and biochemical studies demonstrate that the acyl-enzyme is trapped by the amino-terminal cysteine residue of p35 as an intermediate of trans-thioesterification, similar to that observed in native chemical ligation. Denaturation of a complex between p35 and caspase-8 enabled isolation of the aminoterminus of p35 (residues 2-87) as a cyclic peptide (Lu, et al., Chem. & Biol. (2006) 13:117-22). In this study, we sought to determine whether the cyclic product is sufficient to inhibit caspase-1. Cyclic p35 (2-87) was prepared in situ through the activity of a permuted intein, and caspase activity was monitored through genetic and biochemical screens. We found that the degree of caspase-1 inhibition corresponded to the extent of peptide cyclization, suggesting that cyclic p35 (2-87) is sufficient for caspase inhibition. P35 therefore seems to function as a precursor of a Kunitz-type cyclotide that is matured through caspase cleavage rather than as a Serpin-type suicide substrate inhibitor. (Supported by NIH grant AI053800)