To characterize l-rhamnose isomerase (l-RI) from the thermophilic bacterium Clostridium stercorarium and apply it to produce d-allose from d-allulose. A recombinant l-RI from C. stercorarium exhibited the highest specific activity and catalytic efficiency (k (cat)/K (m)) for l-rhamnose among the reported l-RIs. The l-RI was applied to the high-level production of d-allose from d-allulose. The isomerization activity for d-allulose was maximal at pH 7, 75 A degrees C, and 1 mM Mn2+ over 10 min reaction time. The half-lives of the l-RI at 65, 70, 75, and 80 A degrees C were 22.8, 9.5, 1.9, and 0.2 h, respectively. To ensure full stability during 2.5 h incubation, the optimal temperature was set at 70 A degrees C. Under the optimized conditions of pH 7, 70 A degrees C, 1 mM Mn2+, 27 U l-RI l(-1), and 600 g d-allulose l(-1), l-RI from C. stercorarium produced 199 g d-allose l(-1) without by-products over 2.5 h, with a conversion yield of 33% and a productivity of 79.6 g l(-1) h(-1). To the best of our knowledge, this is the highest concentration and productivity of d-allose reported thus far.