Directed evolution is a proven strategy for generating proteins with improved activities for industrial and therapeutic applications.  A pool of enzyme variants is constructed via mutagenesis and later screened or selected for the desired functions.  Cassette mutagenesis provides a method for targeting contiguous stretches of amino acids for mutation, for example, amino acids in the active site of an enzyme.  Currently, cassette mutagenesis is carried out largely in vitro using the polymerase chain reaction.  Here, we explore the possibility of instead carrying out cassette mutagenesis in vivo, exploiting the efficiency of homologous recombination in S. cerevisiae.  Specifically, we determine the efficiency with which three active site loops in N-(5'-phosphoribosyl)-anthranilate isomerase can be mutagenized based on complementation of a trp1* auxotroph.