Human carbonic anhydrase IV (CA IV) expressed inEscherichia coliwas refolded and activated in cell extracts with the help of endogenous periplasmic protein disulfide isomerase, DsbA, in the presence of oxidized glutathione. The refolding and activation were inhibited by bacitracin but not affected by known cofactors or activators of other chaperones. Although the yield of the purified CA IV recovered from cell extracts was maximal when activated at 4°C in the presence of 2 mmoxidized glutathione, the rate of refolding and activation was much more rapid at 25 and 37°C. The enzyme purified from theE. colicell extracts following activationin vitroshowed similar structural stability and functional properties as CA IV purified from secretion medium from a stably transfected CHO cell line. These studies suggest that the soluble truncated form of human CA IV expressed inE. coli,which is a disulfide-bonded zinc metalloenzyme, can provide a useful model enzyme for studies of protein folding and enzyme activationin vitro.Furthermore, the procedure described for recovery of CA IV following expression inE. colimay be useful forin vitroactivation and subsequent purification of other disulfide-containing proteins.