The growth and buoyant densities of two closely related strains of Escherichia coli in M9-glucose medium that was diluted to produce osmolarities that varied from as low as 5 to 500 mosM were monitored. At 15 mosM, the lowest osmolarity at which buoyant density could be measured reproducibly in Percoll gradients, both ML3 and ML308 had a buoyant density of about 1.079 g/ml. As the osmolarity of the medium was increased, the buoyant density also increased linearly up to about 125 mosM, at which the buoyant density was 1.089 g/ml. From 150 up to 500 mosM, the buoyant density again increased linearly but with a different slope from that seen at the lower osmolarities. The buoyant density at 150 mosM was about 1.091 g/ml, and at 500 mosM it was 1.101 g/ml. Both strains of E. coli could be grown in M9 medium diluted 1:1 with water, with an osmolarity of 120 mosM, but neither strain grew in 1:2-diluted M9 if the cells were pregrown in undiluted M9. (Note: undiluted M9 as prepared here has an osmolarity of about 250 mosM.) However, if the cells were pregrown in 30% M9, about 75 mosM, they would then grow in M9 at 45 mosM and above but not below 40 mosM. To determine which constituent of M9 medium was being diluted to such a low level that it inhibited growth, diluted M9 was prepared with each constituent added back singly. From this study, it was determined that both Ca 2؉ and Mg 2؉ could stimulate growth below 40 mosM. With Ca 2؉ -and Mg 2؉-supplemented diluted M9 and cells pregrown in 75 mosM M9, it was possible to grow ML308 in 15 mosM M9. Strain ML3 would only haltingly grow at 15 mosM. Four attempts were made to grow both ML3 and ML308 at 5 mosM. In three of the experiments, ML308 grew, while strain ML3 grew in one experiment. While our experiments were designed to effect variations in medium osmolarity by using NaCl as an osmotic agent, osmolarity and salinity were changed concurrently. Therefore, from this study, we believe that E. coli might be defined as a euryhalinic and/or euryosmotic bacterium because of its ability to grow in a wide range of salinities and osmolarities.Interest in osmotic regulation of bacteria has had a long and colorful history. Early works of Mitchell (for his review of this early work, see reference 14) led to important discoveries. More recently, interest has been centered more on the physiology of osmotic regulation than the energetics (for a review of more recent work, see reference 8). Epstein and Schultz (10) were able to prove that K ϩ and the counterion glutamate Ϫ played a major role in cytoplasmic osmotic regulation in media of 500 mosM and below (also see a review by Epstein [9]), while the work of Cayley et al. (5, 6) and others (13) has shown that in Escherichia coli betaine and/or proline seems to function as an osmoprotectant for cell growth at higher osmolarities. Some years ago, it was found that the buoyant density of the bacterial cell appeared to be closely related to the osmolarity of the growth medium (2). In those studies, Luria-Bertani medium (LB) supplemented wi...