Bacteria commonly adapt to increases in the osmolarity of the environment by the intracellular accumulation of small organic solutes (osmolytes) that function by restoring turgor. We have investigated the mechanism of osmotic regulation in Rhizobium meliloti, the root nodule symbiont of alfalfa. As a soil microbe, this organism is subjected to variable osmolarity and a number of other environmental conditions to which it must respond appropriately. Natural abundance I3C nuclear magnetic resonance spectroscopy was used to identify the osmolytes accumulated by osmotically stressed R. meliloti. The complement of osmolytes and their concentrations were found to depend on the level of osmotic stress, growth phase of the culture, carbon source, and the presence of osmolytes in the growth medium. The osmolytes accumulated by 22 different bacterial strains are presented and compared to those of R . meliloti 102F34. o