The phosphorylation activities of glucose and levoglucosan (1,6-anhydro-β-d-glucopyranose, LG) were studied in 26 types of LG-assimilating microorganisms isolated from four types of soil in China. The results showed that activities of LG kinase production in most of the yeasts were lower than those of filamentous fungi, and that the highest filamentous fungal activities (0.61 U/mg protein) was approximately twice that of yeast. The filamentous fungi and yeast with highest LG kinase production activity were identified by DNA sequence analysis of 18S or 26S rRNA gene fragments respectively. The identities of them were Alternaria alternata, Eupenicillium javanicum, Aspergillus niger, Penicillium herquei, Cryptococcus laurentii, Cryptococcus flavescens, Cryptococcus luteolus and Rhodotorula aurantiaca, respectively. In addition, the LG kinases from both types of organisms were purified. The effects of temperature and pH on LG kinase activity were also studied. There was very little change in activity between pH 7 to 10 and at temperatures below 30 °C. The apparent Km values for ATP were also similar in different microorganisms, but Km values for LG ranged from 48 to 102 mM.