Glycerol kinase (EC 2.7.1.30; ATP:glycerol 3-phosphotransferase) was purified from Thermus flavus, by ammonium sulfate fractionation and sequential chromatographies on Toyopearl HW65C and DEAE-Toyopearl columns, with an activity recovery of 22.7%. The enzyme is most active at pHs of 9.0 to 9.5. The optimum temperature for the enzyme is 50-70 degrees C. About 50% of the initial activity remains after incubation at 68 degrees C and pH 7.5 for 30 min. The isoelectric point of the enzyme is 4.3. Its molecular weight is estimated to be 220,000 Da by gel filtration on FPLC-Hiload Superdel 200 pg and 58,000 Da by SDS-PAGE, suggesting that it is a tetramer. The activity of the enzyme is completely inhibited by PCMB, HgCl2 and Mn2+. The K-m values of the enzyme for glycerol and ATP are 3.8 x 10(-5) M and 1.62 x 10(-4) M, respectively. The N-terminal amino acid sequence of the enzyme is MNQYMLAIDQGTTSSR.