The effects of salinity and growth temperature on the accumulation of intracellular organic solutes were examined by nuclear magnetic resonance spectroscopy (NMR) in Thermococcus litoralis, Thermococcus celer, Thermococcus stetteri, and Thermococcus zilligii (strain AN1). In addition, the effects of growth stage and composition of the medium were studied in T. litoralis. A novel compound identified as β-galactopyranosyl-5-hydroxylysine was detected in T. litoralis grown on peptone-containing medium. Besides this newly discovered compound, T. litoralis accumulated mannosylglycerate, aspartate, α-glutamate, di-myo-inositol-1,1′(3,3′)-phosphate, hydroxyproline, and trehalose. The hydroxyproline and β-galactopyranosyl-5-hydroxylysine were probably derived from peptone, while the trehalose was derived from yeast extract; none of these three compounds was detected in the other Thermococcus strains examined. Di-myo-inositol-1,1′(3,3′)-phosphate, aspartate, and mannosylglycerate were detected in T. celer and T. stetteri, and the latter organism also accumulated α-glutamate. The only nonmarine species studied, T. zilligii, accumulated very low levels of α-glutamate and aspartate. The levels of mannosylglycerate and aspartate increased in T. litoralis, T. celer, and T. stetteri in response to salt stress, while di-myo-inositol-1,1′(3,3′)-phosphate was the major intracellular solute at supraoptimal growth temperatures. The phase of growth had a strong influence on the types and levels of compatible solutes in T. litoralis; mannosylglycerate and aspartate were the major solutes during exponential growth, while di-myo-inositol-1,1′(3,3′)-phosphate was the predominant organic solute during the stationary phase of growth. This work revealed an unexpected ability of T. litoralis to scavenge suitable components from the medium and to use them as compatible solutes.