The nucleotide sequence of the bglB gene, coding for the thermostable beta-glucosidase B of Clostridium thermocellum was determined. The coding region of 2265 bp was identified by comparison with the N-terminal amino acid sequence of beta-glucosidase B purified from Escherichia coli. The derived amino acid sequence corresponding to a polypeptide of Mr 84,100 was confirmed by sequencing of the C-terminal peptide generated by cleavage with cyanogen bromide. The protein bears no resemblance to other bacterial beta-glucosidase sequences. However, extensive regions of homology were identified between the C. thermocellum enzyme and fungal beta-glucosidases. The N-terminal homologous region contains an amino acid sequence very similar to the active site of beta-glucosidase A3 from Aspergillus wentii. The striking sequence similarities between C. thermocellum beta-glucosidase B and Kluyveromyces fragilis beta-glucosidase suggest the possibility of a genetic exchange between thermophilic anaerobic bacteria and yeasts.