Soybean (Glycine max) grain contains approximately 40% protein and 6.5% nitrogen (N) on an elemental basis. Therefore, the plant requires an abundant N supply throughout its life cycle, and symbiotic N fixation of soybean with Bradyrhizobium japonicum provides 40 to 85% of the soybean N. Although soybean cultivars have been genetically engineered to withstand the herbicide glyphosate, B. japonicum grown in culture is sensitive to glyphosate. We hypothesized that glyphosate applied to glyphosate-tolerant soybean would inhibit nodulation by B. japonicum unless B. japonicum could also be selected for glyphosate tolerance. Cultures of B. japonicum were challenged with sublethal doses of glyphosate, and individual colonies were selected for growth in the presence of glyphosate. Of the 40 isolates that were originally selected for glyphosate tolerance, all isolates in subsequent experiments had similar sensitivity to glyphosate as wild-type B. japonicum. To determine if glyphosate affected B. japonicum in plants, soybean seeds were imbibed with differing levels of glyphosate and water and then planted and inoculated with B. japonicum. After several weeks of growth the plants were harvested and nodules were scanned and analyzed by digital imagery. Glyphosate application to glyphosate-tolerant soybean did not affect the ability of B. japonicum to form nodules and fix nitrogen. These data do not agree with previous responses of small soybean plants sprayed with glyphosate, which showed delayed nodulation and decreased nodule size. It may be that the dosage applied to plants and the timing of the application affect the response of glyphosate on symbiotic effectiveness.