Aspergillus niger is featured with its copious amount of extracellular beta-glucosidase which is generally used to balance the cellulolytic enzyme cocktails for lignocellulose saccharification. However, whether or not A. niger produces any intracellular beta-glucosidase remains obscure. In this study, we analyzed a total of fifteen putative beta-glucosidase genes (bgls) in A. niger CBS 513.88 genome and the five of them were predicted as intracellular bgls due to the lack of signal peptide of extracellular proteins. After further characterization of these five genes through a Saccharomyces cerevisiae in vivo system, only An03g03740 (designated bgl1B) was confirmed to be a beta-glucosidase gene. Western blot and mass spectrometry analysis confirmed BGL1B protein localization inside the cell. BGL1B exhibited the maximal activity at 40 degrees C and pH 5.6. The K-m for p-nitrophenyl-beta-D-glucopyranoside and K-i for glucose were 0.233 +/- 0.058 mM and 119.8 +/- 4.35 mM, respectively. BGL1B showed a strong transglycosylation activity while hydrolyzing cellodextrins with sophorose, laminaribiose, and cellotriose formed from cellobiose, and sophorose and laminaribiose formed from cellotriose. The confirmation of the intracellular beta-glucosidase BGL1B in A. niger further extends our understanding of how A. niger utilizes lignocellulose.