The B-chain of the acidic subunit of crotoxin proved refractory to Edman degradation. When subjected to sequence analysis using tandem mass spectrometry, pyroglutamate was found at the amino-terminal end, even though earlier attempts to de-block with pyroglutamate aminopeptidase were unsuccessful. The B-chain contained 35 amino acids and showed 91% amino acid identity with the corresponding segment from Mojave toxin, a homologous neurotoxin from Crotalus scutulatus scutulatus. The sequence of the last 24 residues of the B-chain is consistent with that previously published (Aird, S.D., Kaiser, I.I., Lewis, R.V. and Kruggel, W.G. (1985) Biochemistry 24, 7054-7058), except at position 20, where Edman degradation gave glycine and mass spectrometry gave glutamic acid.