In higher plants, beta-glucosidases belonging to glycoside hydrolase (GH) Family 1 have been implicated in several fundamental processes including lignification. Phylogenetic analysis of Arabidopsis thaliana GH Family 1 has revealed that At1g61810 (BGLU45), At1g61820 (BGLU46), and At4g21760 (BGLU47) cluster with Pinus contorta coniferin beta-glucosidase, leading to the hypothesis that their respective gene products may be involved in lignification by hydrolysing monolignol glucosides. To test this hypothesis, we cloned cDNAs encoding BGLU45 and BGLU46 and expressed them in Pichia pastoris. The recombinant enzymes were purified to apparent homogeneity by ammonium sulfate fractionation and hydrophobic interaction chromatography. Among natural substrates tested, BGLU45 exhibited narrow specificity toward the monolignol glucosides syringin (K(m), 5.1mM), coniferin (K(m), 7mM), and p-coumaryl glucoside, with relative hydrolytic rates of 100%, 87%, and 7%, respectively. BGLU46 exhibited broader substrate specificity, cleaving salicin (100%), p-coumaryl glucoside (71%; K(m), 2.2mM), phenyl-beta-d-glucoside (62%), coniferin (8%), syringin (6%), and arbutin (6%). Both enzymes also hydrolysed p- and o-nitrophenyl-beta-d-glucosides. Using RT-PCR, we showed that BGLU45 and BGLU46 are expressed strongly in organs that are major sites of lignin deposition. In inflorescence stems, both genes display increasing levels of expression from apex to base, matching the known increase in lignification. BGLU45, but not BGLU46, is expressed in siliques, whereas only BGLU46 is expressed in roots. Taken together with recently described monolignol glucosyltransferases [Lim et al., J. Biol. Chem. (2001) 276, 4344-4349], our enzymological and molecular data support the possibility of a monolignol glucoside/beta-glucosidase system in Arabidopsis lignification.