Three isotoxins (SP I-III) of the beta-bungarotoxin family were purified to homogeneity via a series of isolation procedures including a final step of h.p.l.c. on an SP column washed with a linear gradient of 0.2-0.6 M sodium acetate at pH 7.4. Their proportions varied greatly with the batch of venom. Each isotoxin was demonstrated by SDS/PAGE to contain a phospholipase A2 subunit and a non-phospholipase A2 subunit. The three proteins were reductively alkylated with 4-vinylpyridine and the alkylated derivatives of the two subunits of each isotoxin were separated. N-Terminal sequence analysis of the alkylated derivatives revealed that the three isotoxins probably share a common phospholipase A2 subunit but differ in their non-phospholipase A2 subunits. The non-phospholipase A2 subunits of SP II and SP III were identical with those of beta 2- and beta 1-toxin respectively, except that there was an additional valine inserted between Thr-18 and Val-19 in beta 2-toxin and Pro-18 and Val-19 in beta 1-toxin. The non-phospholipase A2 subunit of SP I differed greatly from that of SP III but was almost identical with that of SP II, except that Lys-14 and Ala-29 in SP II were replaced by Arg-14 and Glu-29 in SP I. Analysis of the effect of CaCl2 on protein fluorescence showed the existence of a low- and a high-affinity site on the different domains of each isotoxin for Ca2+ binding. The three isotoxins showed no great difference in their ability to bind Ca2+ on both the high- and low-affinity site. They had slightly different phospholipase A2 activities but differed to a great extent with respect to their neurotoxic effects. LD50 values increased in the order SP I > SP II > SP III. In contrast, the ability to inhibit the indirectly evoked contraction of chick biventer cervicis muscle was in the order SP III > SP II > SP I.