An alpha-L-fucosidase (E.C. 126.96.36.199) exhibiting a wide aglycon specificity expressed in ability of cleaving alpha1 --> 6-, alpha1 -->3-, alpha1 --> 4-, and alpha1 --> 2-O-fucosyl bonds in fucosylated oligosaccharides, has been isolated from culture filtrate of Thermus sp. strain Y5. The alpha-L-fucosidase hydrolyzes p-nitrophenyl alpha-L-fucopyranoside with V(max) of 12.0 +/- 0.1 microM/min/mg and K(m) = 0.20 +/- 0.05 mM and is able to cleave off about 90% of total L-fucose from pronase-treated fractions of fucosyl-containing glycoproteins and about 30% from the native glycoproteins. The purified enzyme is a tetramer with a molecular mass of 240 +/- 10 kDa consisting of four identical subunits with a molecular mass of 61.0 +/- 0.5 kDa. The N-terminal sequence showed homology to some alpha-L-fucosidases from microbial and plant sources. Hydrolysis of p-nitrophenyl alpha-L-fucopyranoside occurs with retention of the anomeric configuration. Transglycosylating activity of the alpha-L-fucosidase was demonstrated in reactions with such acceptors as alcohols, N-acetylglucosamine and N-acetylgalactosamine while no transglycosylation products were observed in the reaction with p-nitrophenyl alpha-L-fucopyranoside. The enzyme can be classified in glycosyl hydrolase family 29.