Gene duplication with divergence to new functions has been an important mechanism in protein evolution. However, the questions of how many new functions can arise from a particular ancestral gene and how many mutational steps are typically required to generate new functions have been difficult to approach experimentally. We have addressed these questions using T4 lysozyme as a model system by synthesizing two combinatorial libraries of > 10(7) mutant T4 lysozyme genes: one library with an average of 14 missense mutations spread throughout the gene and one library in which 13 active site residues have been simultaneously randomized. These libraries were placed under selection in lacZ or pheA deficient strains of E. coli to investigate whether they sample sufficient diversity to contain mutants with acquired beta-galactosidase or prephenate dehydratase activities. Although neither selection yielded T4 lysozyme mutants with these new activities, a novel E. coli locus was cloned that weakly complements these mutants, allowing them to form 1 mm colonies in 4-6 weeks. This growth rate corresponds to a turnover number of approximately 1000 or 25 min-1 for the lacZ or pheA complementation systems, respectively, thus defining the limits of evolved enzymatic activity detectable in these selections. Thus, the strong selective pressure uncovered an unexpected solution to the biochemical blocks, a frequently observed phenomenon in selection experiments. The characterization of this locus will allow its elimination from future E. coli complementation schemes.