δ-Atracotoxins are novel peptide toxins from the venom of Australian funnel-web spiders that slow sodium current inactivation in a similar manner to scorpion α-toxins. To analyse their interaction with known sodium channel neurotoxin receptor sites we determined their effect on scorpion toxin, batrachotoxin and saxitoxin binding. Nanomolar concentrations of δ-atracotoxin-Hv1 and δ-atracotoxin-Ar1 completely inhibited the binding of the scorpion α-toxin AaH II to rat brain synaptosomes as well as the binding of LqhαIT, a scorpion α-toxin highly active on insects, to cockroach neuronal membranes. Moreover, δ-atracotoxin-Hv1 cooperatively enhanced batrachotoxin binding to rat brain synaptosomes in an analogous fashion to scorpion α-toxins. Thus the δ-atracotoxins represent a new class of toxins which bind to both mammalian and insect sodium channels at sites similar to, or partially overlapping with, the receptor binding sites of scorpion α-toxins.