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The venom of the European black widow spider Latrodectus tredecimguttatus (Theridiidae) contains several high molecular mass (110–140 kDa) neurotoxins that induce neurotransmitter exocytosis. These include a vertebrate-specific α-latrotoxin (α-LTX-Lt1a) responsible for the clinical symptoms of latrodectism and numerous insect-specific latroinsectoxins (LITs). In contrast, little is known about the expression of these toxins in other Latrodectus species despite the fact that envenomation by these spiders induces a similar clinical syndrome. Here we report highly conserved α-LTX, α-LIT and δ-LIT sequence tags in Latrodectus mactans, Latrodectus hesperus and Latrodectus hasselti venoms using tandem mass spectrometry, following bioassay-guided separation of venoms by liquid chromatography. Despite this sequence similarity, we show that the anti-α-LTX monoclonal antibody 4C4.1, raised against α-LTX-Lt1a, fails to neutralize the neurotoxicity of all other Latrodectus venoms tested in an isolated chick biventer cervicis nerve–muscle bioassay. This suggests that there are important structural differences between α-LTXs in theridiid spider venoms. We therefore cloned and sequenced the α-LTX from the Australian red-back spider L. hasselti (α-LTX-Lh1a). The deduced amino acid sequence of the mature α-LTX-Lh1a comprises 1180 residues (∼132 kDa) with ∼93% sequence identity with α-LTX-Lt1a. α-LTX-Lh1a is composed of an N-terminal domain and a central region containing 22 ankyrin-like repeats. The presence of two furin cleavage sites, conserved with α-LTX-Lt1a, indicates that α-LTX-Lh1a is derived from the proteolytic cleavage of an N-terminal signal peptide and C-terminal propeptide region. However, we show that α-LTX-Lh1a has key substitutions in the 4C4.1 epitope that explains the lack of binding of the monoclonal antibody.