The possibility of raising a humoral immune response capable of inducing in vivo protection against the lethal effects of Tityus serrulatus (Ts) scorpion venom was evaluated in the mouse model. An immunogen was prepared that consists of a toxic fraction (TstFG50) of the Tityus venom (this G 50 chromatography fraction represents most of the toxicity of the crude venom) conjugated to bovine serum albumin (BSA) with glutaraldehyde. TstFG50 coupled to BSA yielded a thoroughly detoxified immunogen. BALB/c and C57BL/10 mice were immunized with this preparation and all developed an antibody response. In vivo protection assays one week after the last immunization showed that vaccinated mice could resist the challenge by twice the LD50 of the TstFG50, a dose which killed all control non-immune mice. The protective effect persisted nine weeks after the end of the immunization protocol. To characterize epitopes of protective antibodies we used the Spot method of multiple peptide synthesis to prepare sets of immobilized 15mer overlapping peptides, covering the complete amino acid sequences of the main Tityus toxins, TsII and TsVII (both β-type toxins) and TsIV, an α-type toxin that is the major lethal component of the venom. Antibody binding to peptides, revealed one major antigenic region in the C-terminal part of the three toxins and another region in the helical part of TsII and TsIV toxins. It is likely that these epitopes correspond to neutralizing epitopes since they correspond to regions of the toxins that are known to be involved in the active site of the toxins. © 2004 Elsevier Ltd. All rights reserved.