In this study we propose revised structures for the two major compatible solutes of Rhodothermus marinus. We have also examined the accumulation of compatible solutes by the type strains of the slightly halophilic and thermophilic species Rhodothermus marinus and Rhodothermus obamensis at several growth temperatures and salinities. The major solutes of R. marinus were identified as alpha-mannosylglycerate (alpha-MG) and alpha-mannosylglyceramide (alpha-MGA), whereas R. obamensis accumulated only alpha-mannosylglycerate. The total osmolyte content was higher during the early exponential phase and decreased abruptly as growth continued into the stationary phase. At low growth temperatures. R. marinus responded to water stress by accumulation of alpha-mannosylglycerate and its amide, in addition to low levels of trehalose, glutamate, and glucose. At the highest growth temperature, alpha-mannosylglycerate was the major compatible solute and alpha-mannosylglyceramide was not detected. When both compounds were present, an increase in the salinity of the growth medium favored the accumulation of alpha-mannosylglyceramide over alpha-mannosylglycerate. The absence of alpha-mannosylglyceramide in R. obamensis at all growth temperatures and salinities constituted the most pronounced difference in the profiles of compatible solute accumulation by the two strains. Trehalose was also a prominent solute in this organism. Both organisms accumulated higher levels of alpha-mannosylglycerate as the temperature was raised. The importance of the two compounds in the mechanisms of thermoadaptation and osmoadaptation is discussed.