Glyphosate or Roundup is the most extensively used herbicide for broad-spectrum control of weeds. Glyphosate inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a key enzyme in the aromatic amino acid biosynthetic pathway in microorganisms and plants. Applying the staggered extension process, we randomly mutated and recombined the aroA genes of Salmonella typhimurium and Escherichia coli to obtain four variants that exhibit significantly enhanced tolerance to glyphosate. All four mutants are chimeras of the two parental genes and, in addition, three of them carry one or more de novo point mutations. None of the amino acid substitutions in the mutants was in a position previously known to be important for catalysis or substrate binding. Kinetic analysis of EPSPS activity from these mutants indicated that the tolerance was attributed to a 2-10-fold increased specific activity, 0.4-8-fold reduced affinity to glyphosate, and 2.5-19-fold decreased K(m) for phosphoenolpyruvate. Such mutants will be instrumental for the structural and function study of the enzyme and for the generation of transgenic crops resistant to the herbicide.