The thermophilic and halotolerant nature of Rubrobacter xylanophilus led us to investigate the accumulation of compatible solutes in this member of the deepest lineage of the Phylum Actinobacteria. Trehalose and mannosylglycerate (MG) were the major compounds accumulated under all conditions examined, including those for optimal growth. The addition of NaCl to a complex medium and a defined medium had a slight or negligible effect on the accumulation of these compatible solutes. Glycine betaine, di-myo-inositol-phosphate (DIP), a new phosphodiester compound, identified as di-N-acetyl-glucosamine phosphate and glutamate were also detected but in low or trace levels. DIP was always present, except at the highest salinity examined (5% NaCl) and at the lowest temperature tested (43 degrees C). Nevertheless, the levels of DIP increased with the growth temperature. This is the first report of MG and DIP in an actinobacterium and includes the identification of the new solute di-N-acetyl-glucosamine phosphate.