Although only five isotoxins of the beta-bungarotoxin family had been claimed, this work indicated the existence of more than sixteen isotoxins. Crude snake (Bungarus multicinctus) venom was divided into four main fractions by gel filtration on a Sephadex G-50 column. beta-Bungarotoxin appeared in a major fraction that contained mainly the M(r) 20,000 protein components. The fraction could be further resolved into eighteen peaks designated P1-P18 by HPLC on a Protein Pak SP 5PW column that was eluted with a linear gradient of 0.1-0.6 M CH3COONa in 20 mM NaH2PO4-Na2HPO4 at pH 7.4 P3-P18 were demonstrated to be isotoxins of the beta-bungarotoxin family. Results of protein sequencing for P8, P9 and P11, the three main isotoxins, confirmed that they shared a common phospholipase A2 subunit, which was very similar to although not completely identical with the A1 chain reported previously.