The folding of hirudin undergoes an initial stage of non-specific packing, followed by consolidation (re-organization) of partially packed intermediates to attain the native structure [Chatrenet and Chang (1993) J. Biol. Chem. 268, 20988-20996]. Non-specific packing leads to the formation of scrambled hirudins as folding intermediates. A systematic study was carried out to search for conditions which would selectively control and enhance the processes of packing and consolidation. It is demonstrated here that under optimized conditions, including the use of cystine/cysteine and protein disulphide isomerase, the folding of hirudin in vitro can be achieved quantitatively within 30 s.