In this report, all of the criteria necessary for the demonstration of nitrogen fixation in wheat (Triticum aestivum L.), the world's most important crop, are shown upon inoculation with a nitrogen-fixing bacterium, Klebsiella pneumoniae 342 (Kp342). Kp342 relieved nitrogen (N) deficiency symptoms and increased total N and N concentration in the plant. Nitrogen fixation was confirmed by 15N isotope dilution in the plant tissue and in a plant product, chlorophyll. All of these observations were in contrast to uninoculated plants, plants inoculated with a nitrogen-fixing mutant of Kp342, and plants inoculated with dead Kp342 cells. Nitrogenase reductase was produced by Kp342 in the intercellular space of the root cortex. Wild-type Kp342 and the nifH mutant colonized the interior of wheat roots in equal numbers on a fresh weight basis. The nitrogen fixation phenotype described here was specific to cv. Trenton. Inoculation of cvs. Russ or Stoa with Kp342 resulted in no relief of nitrogen deficiency symptoms.