The target site of glyphosate [N-(phosphonomethyl)glycine] inhibition in plants and bacteria is 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase. Our strategy for developing glyphosate-resistant crops has been to genetically engineer plants with a gene that codes for EPSP synthase with low sensitivity in glyphosate. We cloned such a gene from the aroA locus of a glyphosate-resistant mutagenized strain of Salmonella typhimurium. The enzyme encoded by this gene has a single amino acid change resulting in lower affinity for glyphosate and higher affinity for substrates than either plant or wild-type bacterial counterpart. A chimaeric gene containing the mutant aroA gene behind the octopine synthase promoter was constructed and integrated into Agrobacterium T-DNA vectors. Analysis of gall tissue from Brassica campestris L. (turnip rape) infected with A. tumefaciens K12 containing this chimaera showed mRNA and protein expressed from the bacterial gene; 50% of the total EPSP synthase activity present had kinetic properties of the mutant bacterial enzyme. Tobacco (Nicotiana tabacum L. ‘Xanthi′) plants have been regenerated from cocultivation with A. rhizogenes containing the same construct; analysis indicates expression of the gene and enhanced tolerance to glyphosate.