A mass spectrometric method was applied to the B subunit of Vibrio cholerae classical biotype Inaba 569B toxin to determine its amino acid sequence and to confirm the differences in the amino acid sequences predicted from the nucleotide sequences of the genes of El Tor biotype strains 62746 and 2125 toxins. In this method, the Staphylococcus aureus protease V8 digest of the CNBr-treated B subunit of the classical biotype toxin was examined directly by fast-atom-bombardment mass spectrometry without separation of individual peptides. The values of molecular ion signals observed in the mass spectra were compared with the amino acid sequences of the classical biotype and El Tor biotype toxins. All the observed mass values coincided with those calculated from the published sequences of the B subunit except those of the sequences at positions 12-29 and 69-79. Peptides with these sequences were isolated by high-performance liquid chromatography and analyzed by Edman degradation or by combination of mass spectrometry and enzymatic degradation. The results revealed that the amino acid residues at positions 22 and 70 were Asp instead of Asn in the published sequences of classical biotype toxin. It was also found that Asn at position 44 was partially deaminated to Asp. The amino acid sequence of the classical biotype toxin was found to be different only at positions 18 (His----Tyr), 47 (Thr----Ile) and 54 (Gly----Ser) from that of El Tor biotype toxins.