Four strains of Rhizobium japonicum (61A76 and USDA 110, 123, and 138) were grown in eight different defined media. Regardless of the carbon or nitrogen source supplied, alpha, alpha-trehalose was the major carbohydrate (among mono- and disaccharides) accumulated by all four strains. After 7 to 9 days of growth, trehalose generally accounted for 90 to 100% of the mono- and disaccharides detected. None of the four strains would grow with trehalose as a carbon source, but the utilization of endogenous trehalose was demonstrated under carbon starvation conditions in water culture or when the carbon supply in a defined medium was exhausted. Under these conditions, a small amount of trehalose was lost from cells to the medium. In a survey of most of the serogroups of R. japonicum and several strains of other Rhizobium species, all strains tested were found to accumulate some trehalose. Trehalose concentrations varied widely; the highest concentration recorded was 41 micrograms/mg of dry weight. In all but six strains trehalose accounted for greater than 80% of the mono- and disaccharides in cells. Fast-growing strains of R. japonicum also accumulated small amounts trehalose. R. japonicum bacteroids also synthesized trehalose; the quantity in nodules varied in approximate correspondence to accumulation of trehalose by cultured bacteria. In young soybean nodules (29 days after planting), 45 to 80% of the trehalose was recovered in the cytosol. There were differences among R. japonicum strains in the retention of trehalose, and the proportion of trehalose retained by bacteroids increased with increasing plant age for all strains.