Methanococcus igneus, a hyperthermophilic marine methanogen (optimum growth temperature of 88°C) with a 25-min doubling time, synthesizes an unusual inositol phosphodiester which is present at high intracellular concentrations along with l-α-glutamate and β-glutamate. Identification of this compound as a dimeric inositol phosphodiester (di-myo-inositol-1,1′-phosphate) was provided by two-dimensional nuclear magnetic resonance methods. The intracellular levels of all three negatively charged solutes (l-α-glutamate, β-glutamate, and the inositol phosphodiester) increase with increasing levels of external NaCl, although the inositol compound shows much smaller increases with increasing NaCl levels than the glutamate isomers. The turnover of these solutes was examined by 13CO2-pulse-CO2-chase experiments. The results indicated that both the β-glutamate and the inositol phosphodiester behaved as compatible solutes and were not efficiently metabolized by cells as was l-α-glutamate. At a fixed external NaCl concentration, lower ammonium levels increased the fraction of the inositol dimer present in extracts. The most pronounced changes in di-myo-inositol-1,1′-phosphate occurred as a function of cell growth temperature. While the organism grows over a relatively wide temperature range, the phosphodiester accumulated only when M. igneus was grown at temperatures of ≥80°C. Thus, this unusual compound is a non-nitrogen-containing osmolyte preferentially synthesized at high growth temperatures.