Glyphosate (N-phosphonomethyl-glycine) is the active ingredient in the nonselective herbicide Roundup. The sensitivity of crop plants to glyphosate has limited its in-season use as a postemergence herbicide. The extension of the ue of Roundup herbicide to allow in-season application in major crops such as soybeans [Glycine max (L.) Merr.] would provide new weed control options for farmers. A glyphosate-tolerant soybean line, 40-3-2, was obtained through expression of the bacterial 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase, EPSPS) enzyme from Agrobacterium sp. strain CP4. Line 40-3-2 is highly tolerant to glyphosate, showing no visual injury after application of up to 1.68 kg acid equivalent (a.e.) ha-1 of glyphosate under field conditions. Molecular characterization studies determined that the single genetic insert in line 40-3-2 contains only a portion of the cauliflower mosaic virus 35S promoter (P-E35S), the Petunia hybrida EPSPS chloroplast transit peptide (CTP), the CP4 EPSPS gene, and a portion of the 3' nontranslated region of the nopaline synthase gene (NOS3') terminator. Inheritance studies have shown that the transgene behaves as a single dominant gene and is stable over several generations.