The complete nucleotide sequence of the -glucosidase gene of Kluyveromyces fragilis has been determined. This sequence contains an open reading frame of 2535 base pairs encoding a protein of 845 amino acids. Analysis of the transcription products revealed only one transcript of about 3 kb identical in both Kluyveromyces fragilis and in the expression host Saccharomyces cerevisiae. The protein molecular weight of 93,811 Kd deduced from the sequence is consistent with the 90,000 Kd determined by SDS polyacrylamide gel electrophoresis with the purified protein. Mapping of the starts of transcription shows that two starting points are used in the natural host Kluyveromyces fragilis. A comparison of the amino acid sequence with that of other -glucosidases revealed three regions of homology. One of these regions contains an amino acid sequence very similar to a peptide isolated from the active site of -glucosidase A₃ from Aspergillus wentii and could be implicated in the catalytic mechanism of these glucolytic enzymes.