An α-l-arabinofuranosidase of GH62 from Aspergillus nidulans FGSC A4 ( An Abf62A-m2,3) has an unusually high activity towards wheat arabinoxylan (WAX) (67 U/mg; k cat = 178/s, K m = 4.90 mg/ml) and arabinoxylooligosaccharides (AXOS) with degrees of polymerisation (DP) 3–5 (37–80 U/mg), but about 50 times lower activity for sugar beet arabinan and 4-nitrophenyl-α-l-arabinofuranoside. α-1,2- and α-1,3-linked arabinofuranoses are released from monosubstituted, but not from disubstituted, xylose in WAX and different AXOS as demonstrated by NMR and polysaccharide analysis by carbohydrate gel electrophoresis (PACE). Mutants of the predicted general acid (Glu 188 ) and base (Asp 28 ) catalysts, and the general acid p K a modulator (Asp 136 ) lost 1700-, 165- and 130-fold activities for WAX. WAX, oat spelt xylan, birchwood xylan and barley β-glucan retarded migration of An Abf62A-m2,3 in affinity electrophoresis (AE) although the latter two are neither substrates nor inhibitors. Trp 23 and Tyr 44 , situated about 30 Å from the catalytic site as seen in an AnAbf62A-m2,3 homology model generated using Streptomyces thermoviolaceus Sth Abf62A as template, participate in carbohydrate binding. Compared to wild-type, W23A and W23A/Y44A mutants are less retarded in AE, maintain about 70 % activity towards WAX with K i of WAX substrate inhibition increasing 4–7-folds, but lost 77–96 % activity for the AXOS. The Y44A single mutant had less effect, suggesting Trp 23 is a key determinant. AnAbf62A-m2,3 seems to apply different polysaccharide-dependent binding modes, and Trp 23 and Tyr 44 belong to a putative surface binding site which is situated at a distance of the active site and has to be occupied to achieve full activity.