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Among a few cell lines of maize (Zea mays L., cv. Black Mexican Sweet (MS)) tested, one showed a natural, remarkable tolerance to glyphosate at concentrations as high as 10 mM. Two activities of 5-enol-pyruvyl-shikimate-3-phosphate (EPSP) synthase, the target enzyme of the herbicide, were separated in the tolerant culture by anion-exchange chromatography. One peak of activity was not significantly inhibited by glyphosate even at millimolar concentrations. While the glyphosate-sensitive isoform persisted throughout the growth cycle of the culture, the glyphosate-resistant EPSP synthase increased only after the onset of exponential growth and declined in the stationary phase. Tolerance to the herbicide was accompanied by a reduced affinity of the enzyme for the substrate phospho-enol-pyruvic acid.