Venoms from Tityus species inhabiting five endemic regions of scorpionism in Venezuela (Andean, Perijá range, north-central, northeastern, and Guayana) and also southeast Brazil (T. serrulatus and T. bahiensis) were characterized immunologically in ELISA experiments using mouse- and rabbit-derived antibodies to evaluate their cross-reactivity and also functionally, utilizing synaptosome binding assays. While Brazilian and Venezuelan antivenoms cross-reacted poorly, T. discrepans (north-central Venezuela) and T. zulianus (Andean) venoms shared a greater immunological relatedness than with T. perijanensis (Perijá range). Anti-T. breweri (Guayana) antibodies fully cross-reacted with T. discrepans. Native PAGE indicated species-specific fingerprints for all venoms and revealed differences between two populations (Anzoátegui and Monagas States) of T. nororientalis (northeastern Venezuela). Components antigenically related to T. serrulatus β-toxin TsVII were also detected in T. breweri, T. nororientalis (Anzoátegui) and T. funestus (Andean). Antibodies against T. serrulatus anatoxin TsNTxP did not cross-react significantly with any Venezuelan venoms indicating lack of TsNTxP homologues. The results suggest that the extent of antigenic reactivity depends on the studied species rather than the geographical distance between their habitats. All venoms, with T. discrepans to a lesser extent, were able to significantly displace [125I]-TsVII from its binding site in rat brain synaptosomes. Our data indicate that β-toxins functionally related to TsVII but differing significantly in their antigenic regions exist in Venezuelan venoms from different endemic regions. Identification of shared epitopes with TsVII, at least for some species, may lead to the design of antibodies based on common epitopes for treating scorpion envenoming in Venezuela and Brazil. © 2007 Elsevier Ltd. All rights reserved.