The broad-spectrum, non-selective herbicide glyphosate [N-(phosphonomethyl)glycine] is a potent inhibitor of highly purified 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (3-phosphoshikimate 1-carboxyvinyltransferase, EC 2.5.1.19) of Klebsiella pneumoniae. The inhibition is competitive with phosphoenolpyruvate (PEP) with Ki = 1 microM at pH 6.8 and non-competitive with shikimate 3-phosphate, EPSP, and inorganic phosphate. Non-herbicidal analogues of glyphosate, such as aminomethylphosphonic acid, bis-N-(phosphonomethyl)glycine and iminodiacetic acid, do not inhibit the enzyme. Inhibition of EPSP synthase by glyphosate strongly increases with increasing pH. Glyphosate protects the enzyme against inactivation by phenylglyoxal, 3-bromopyruvate, and N-ethylmaleimide. It is proposed that glyphosate binds to the PEP-binding site of EPSP synthase as a transition-state analogue of PEP. Other PEP-utilizing enzymes were not found to be subject to inhibition by glyphosate.