A family 51 arabinoxylan arabinofuranohydrolase, designated AXAH-I, has been purified from extracts of 7-day-old barley (Hordeum vulgare L.) seedlings by fractional precipitation with (NH(4))(2)SO(4) and ion-exchange chromatography. The enzyme has an apparent molecular mass of 65 kDa and releases L-arabinose from cereal cell wall arabinoxylans with a pH optimum of 4.3, a catalytic rate constant (k(cat)) of 6.9 s(-1) and a catalytic efficiency factor (k(cat)/K(m)) of 0.76 (ml x s(-1) x mg(-1)). Whereas the hydrolysis of alpha-L-arabinofuranosyl residues linked to C(O)3 of backbone (1-->4)-beta-xylosyl residues proceeds at the fastest rate, alpha-L-arabinofuranosyl residues on doubly substituted xylosyl residues are also hydrolysed, at lower rates. A near full-length cDNA encoding barley AXAH-I indicates that the mature enzyme consists of 626 amino acid residues and has a calculated pI of 4.8. A second cDNA, which is 81% identical with that encoding AXAH-I, encodes another barley AXAH, which has been designated AXAH-II. The barley AXAHs are likely to have key roles in wall metabolism in cereals and other members of the Poaceae. Thus the enzymes could participate in the modification of the fine structure of arabinoxylan during wall deposition, maturation or expansion, or in wall turnover and the hydrolysis of arabinoxylans in germinated grain.