BACKGROUND:  Glyphosate tolerance is a dominant trait in modern biotech crops.RESULTS:  A gene encoding a glyphosate-tolerant EPSP synthase (aroA1398) from bacterial strain ATX1398 was cloned and characterized.  The protein is initiated at a GTG translational start codon to produce a protein that provides robust glyphosate resistance in Escherichia coli (Mig) Cast & Chalm.  The aroA1398 protein was expressed and purified from E. coli, and key kinetic values were determined (Ki = 161 µM; Km(PEP) = 11.3 µM; kcat = 28.3 s-1).  The full-length enzyme is 800-fold more resistant to glyphosate than the maize EPSP synthase while retaining high affinity for the substrate phosphoenol pyruvate.  To evaluate further the potential of aroA1398, transgenic maize events expressing the aroA1398 protein were generated.  T0 plants were screened for tolerance to glyphosate sprays at 1.3× commercial spray rates, and T1 plants were selected that completely resisted glyphosate sprays at 1×, 2× and 4× recommended spray rates in field trials.CONCLUSION:  These data suggest that aroA1398 is a suitable candidate for conferring glyphosate tolerance in transgenic crop plants.