A non-toxic protein (TsNTxP) isolated from the venom of the noxious scorpion Tityus serrulatus (Ts) induces polyclonal antibodies cross-reactive with several toxins from the venom, in sharp contrast to anti-toxin antibodies which are toxin specific. To try to uncover the molecular basis for these unusual properties, peptide scanning experiments were performed and indicated that the N- and C-terminal parts of TsNTxP enclose continuous epitopes (residues 1-15 and 47-61). Antibodies raised against peptides corresponding to these two regions were found to have neutralizing properties against a mixture of all toxic proteins from the T. serrulatus venom, indicating that residues 1-15 and 47-61 correspond to neutralizing epitopes. The identification of key antigenic residues within these two epitopes revealed that several of them are well conserved in the amino-acid sequences of the three main toxins (Ts II, Ts IV and Ts VII) from the venom: Glu 3, Tyr 5, Asp 8, Asp 50, Trp 55 and Lys 61. A single key-residue (Glu 58) is unique to TsNTxP. By using homology modeling, a model of the three-dimensional structure of TsNTxP was obtained. The antigenically important residues from TsNTxP were found to be surface exposed, with five of them clustered on the facet of the protein reported to enclose the active site of toxins. Residues equivalent to the seven key-residues of the anatoxin were also found to be exposed in the active toxins from T. serrulatus venom. These results show that antibodies elicited by the non-toxic protein TsNTxP recognized, within the N- and C-terminal parts of toxins of T. serrulatus, conserved and surface exposed residues which might also be involved in the toxic action of the proteins. © 2002 Elsevier Science Ltd. All rights reserved.