Cytochrome c peroxidase (CCP) from Saccharomyces cerevisiae was subjected to directed molecular evolution to generate mutants with increased activity against 2,2 ' -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). Using a combination of DNA shuffling and saturation mutagenesis, mutants were isolated which possessed more than 20-fold increased activity against ABTS and a 70-fold increased specificity toward ABTS compared to the natural substrate. In contrast, activities against another small organic molecule, guaiacol, were not significantly affected. Mutations at residues Asp224 and Asp217 were responsible for this increase in activity. These two residues are located on the surface of the protein and not in the direct vicinity of the distal cavity of the peroxidase, where small organic substrates are believed to be oxidized. Mutations at position Asp224 also lead to an increased amount of the active holoenzyme expressed in Escherichia coli, favoring the selection of these mutants in the employed colony screen. Possible explanations for the effect of the mutations on the in vitro activity of CCP as well as the increased amount of holoenzyme are discussed.