The neurotoxicity observed following death adder envenoming has been thought to be solely due to the presence of potent post-synaptic neurotoxins. Clinically, these effects are often poorly reversed by death adder antivenom or anticholinesterase, particularly when patients present with established paralysis. This suggests that either the post-synaptic neurotoxins are irreversible/ pseudo irreversible, or the venom contains pre-synaptic neurotoxins that do not respond to antivenom. To support the later hypothesis, a pre-synaptic neurotoxin (P-EPTX-Aa1a) has recently been isolated from the venom of Acanthophis antarcticus. We examined A. praelongus and A. rugosus venoms for the presence of pre-synaptic neurotoxins. P-EPTX-Ap1a (40,719 Da) and P-EPTX-Ar1a (40,879 Da) were isolated from A. praelongus and A. rugosus venoms, respectively. P-EPTX-Ap1a and P-EPTX-Ar1a are comprised of three different subunits, alpha, beta1 and beta2. The two toxins displayed similar levels of PLA(2) activity which was almost solely attributed to the alpha subunit in both toxins. P-EPTX-Ap1a (20-100nM) and P-EPTX-Ar1a (20-100nM) caused inhibition of indirect twitches of the skeletal muscle preparation without affecting contractile responses to nicotinic receptor agonists. Interestingly, only the alpha subunit of both toxins (300nM) displayed neurotoxic activity. Inhibition of PLA(2) activity markedly reduced the effect of the toxins on muscle twitch height. These results confirm that P-EPTX-Ap1a and P-EPTX-Ar1a are pre-synaptic neurotoxins and represent the second and third such toxins to be isolated from death adder venom. The presence of pre-synaptic neurotoxins in Acanthophis sp. venoms indicates that treatment strategies for envenoming by these snakes needs to be reassessed given the likelihood of irreversible neurotoxicity.