When the "suicide" vector pSUP1011, which carries transposon Tn5 (Kmr), was introduced into Rhizobium japonicum USDA 110, kanamycin-resistant (Kmr) colonies were detected at a frequency (4.2 X 10-6) ca. 30 times greater than the spontaneous kanamycin resistance frequency (1.4 X 10-7). Ten thousand Kmr mutants were isolated and tested for nutritional auxotrophy. Auxotrophs were detected at a frequency of 0.5%. The following classes of auxotrophs were identified: adenine- (three), histidine- (three), glutamate- (five), adenine plus thiamine- (nine), uracil- (three), pantothenic acid- (one), tryptophan- (three), and methionine- (three). Mutants blocked in symbiotic nitrogen fixation (Fix-) were also identified at a frequency of 3%. The glutamate auxotrophs were studied in more detail, and all five showed an altered expression of nitrogenase activity in free-living cultures.