Polyclonal antibodies raised against a synthetic peptide (sequence 50-59) of Androctonus australis Hector toxin II can neutralize the effects of toxin II in vivo. The antigenic specificities of anti-peptide and anti-toxin antibodies were compared by competitive aqueous phase radioimmunoassay by using 125I-toxin II, chemically modified or homologous toxins, and the synthetic peptide 50-59, either free or bound to bovine serum albumin (BSA). The antipeptide and anti-toxin antibodies had a comparable high affinity for the native toxin, but anti-peptide antibodies exhibited a lower binding capacity. Anti-peptide antibodies had a higher affinity for native toxin than for the peptide 50-59 bound to BSA, used as immunogen, and were unable to recognize the free peptide. These results suggest that it is necessary to restrict the conformational freedom of the immunizing peptide in order to obtain anti-peptide antibodies with a high affinity for the toxin. The lysine residue at position 58 of toxin II, essential for toxicity, appears to be immunogenic when immunization is with peptide 50-59 bound to BSA and not with the native toxin. This residue is antigenic in the native toxin, however, as shown by the anti-peptide antibodies.