Most of the "repressor, open reading frame, kinase" (ROK) proteins already characterized so far, and exhibiting a kinase activity, take restrictedly d-glucose as substrate. By exploring the sequenced bacterial diversity, 61 ATP-dependent kinases belonging to the ROK family have been identified and experimentally assayed for the phosphorylation of hexoses. These kinases were mainly found to be thermotolerant and highly active toward d-mannose and d-fructose with notable activities toward d-tagatose. Among them, the ATP-dependent kinase from the mesophile Streptococcus mitis (named ScrK(mitis)) was biochemically characterized and its substrate spectrum further studied. This enzyme possessed impressive catalytic efficiencies toward d-mannose and d-fructose of 1.5 10(6) s(-1) M-1 and 2.7 10(5) s(-1) M-1, respectively, but also significant ones toward d-tagatose (3.5 10(2) s(-1) M-1) and the unnatural monosaccharides d-altrose (1.1 10(4) s(-1) M-1) and d-talose (3.4 10(2) s(-1) M-1). Specific activities measured for all hexoses showed a high stereopreference for d- over l-series. As proof of concept, 8 hexoses were phosphorylated in moderate to good yields, some of them described for the first time like l-sorbose-5-phosphate unusually phosphorylated in position 5. Its thermotolerance, its wide pH tolerance (from 7 to 10), and temperature range (> 85% activity between 40 and 70 A degrees C) open the way to applications in the enzymatic synthesis of monophosphorylated hexoses.