Aims: To characterize of a thermostable recombinant alpha-l-arabinofuranosidase from Caldicellulosiruptor saccharolyticus for the hydrolysis of arabino-oligosaccharides to l-arabinose. Methods and Results: A recombinant alpha-l-arabinofuranosidase from C. saccharolyticus was purified by heat treatment and Hi-Trap anion exchange chromatography with a specific activity of 28 center dot 2 U mg-1. The native enzyme was a 58-kDa octamer with a molecular mass of 460 kDa, as measured by gel filtration. The catalytic residues and consensus sequences of the glycoside hydrolase 51 family of alpha-l-arabinofuranosidases were completely conserved in alpha-l-arabinofuranosidase from C. saccharolyticus. The maximum enzyme activity was observed at pH 5 center dot 5 and 80 degrees C with a half-life of 49 h at 75 degrees C. Among aryl-glycoside substrates, the enzyme displayed activity only for p-nitrophenyl-alpha-l-arabinofuranoside [maximum k(cat)/K-m of 220 m(mol l-1)-1 s-1] and p-nitrophenyl-alpha-l-arabinopyranoside. This substrate specificity differs from those of other alpha-l-arabinofuranosidases. In a 1 mmol l-1 solution of each sugar, arabino-oligosaccharides with 2-5 monomer units were completely hydrolysed to l-arabinose within 13 h in the presence of 30 U ml-1 of enzyme at 75 degrees C. Conclusions: The novel substrate specificity and hydrolytic properties for arabino-oligosaccharides of alpha-l-arabinofuranosidase from C. saccharolyticus demonstrate the potential in the commercial production of l-arabinose in concert with endoarabinanase and/or xylanase. Significance and Impact of the Study: The findings of this work contribute to the knowledge of hydrolytic properties for arabino-oligosaccharides performed by thermostable alpha-l-arabinofuranosidase.