Serpins are a superfamily of structurally conserved proteins. Inhibitory serpins use a suicide substrate-like mechanism. Some are able to inhibit cysteine proteases in cross-class inhibition. Here, we demonstrate for the first time the strong inhibition of initiator and effector caspases 3 and 8 by two purified bovine SERPINA3s. SERPINA 3-1 (uniprotkb:Q9TTE1) binds tighly to human CASP3 (uniprotkb:P42574) and CASP8 (uniprotkb:Q14790) with k(ass) of 4.2x10(5) and 1.4x10(6) M(-1)s(-1), respectively. A wholly similar inhibition of human CASP3 and CASP8 by SERPINA3-3 (uniprotkb:Q3ZEJ6) was also observed with k(ass) of 1.5x10(5) and 2.7x10(6) M(-1)s(-1), respectively and form SDS-stable complexes with both caspases. By site-directed mutagenesis of bovSERPINA3-3, we identified Asp(371) as the potential P1 residue for caspases. The ability of other members of this family to inhibit trypsin and caspases was analysed and discussed.