The genes encoding pro-carboxypeptidase Ys (pro-CPYs) from Saccharomyces cerevisiae and Hansenula polymorpha were cloned and expressed in Escherichia coli. Most of the expressed pro-CPY was present in the form of inclusion bodies, accounting for about 35% of the total cellular protein. The inclusion bodies solubilized in 6 M guanidinum chloride were renatured by dilution 1:60 into renaturation buffer. Further refolding and activation were followed by the addition of 60 μg ml-1 proteinase K into the diluted solution. The specific activities of in vitro activated S. cerevisiae and H. polymorpha CPYs were found to be similar, representing about 25% of that of native S. cerevisiae CPY.