The metastability of inhibitory serpins (serine proteinase inhibitors) is thought to play a key role in the facile conformational switch and the insertion of the reactive center loop into the central β-sheet, A-sheet, during the formation of a stable complex between a serpin and its target proteinase. We have examined the folding and inhibitory activity of a very stable variant of human α1-antitrypsin, a prototype inhibitory serpin. A combination of seven stabilizing single amino acid substitutions of α1- antitrypsin, designated Multi-7, increased the midpoint of the unfolding transition to almost that of ovalbumin, a non-inhibitory but more stable serpin. Compared with the wild-type α1-antitrypsin, Multi-7 retarded the opening of A-sheet significantly, as revealed by the retarded unfolding and latency conversion of the native state. Surprisingly, Multi-7 α1- antitrypsin could form a stable complex with a target elastase with the same kinetic parameters and the stoichiometry of inhibition as the wild type, indicating that enhanced A-sheet closure conferred by Multi-7 does not affect the complex formation. It may be that the stability increase of Multi-7 α1- antitrypsin is not sufficient to influence the rate of loop insertion during the complex formation.