Bites by Australian black snakes (Pseudechis spp.) do not cause neurotoxicity in human envenoming. This is unusual as in vitro neurotoxicity has been reported for all Pseudechis spp. venoms. The present study aimed to identify, isolate and characterise neurotoxins from the venoms of Pseudechis porphyriacus and Pseudechis colletti to elucidate the reason for the lack of neurotoxicity in humans. alpha-Elapitoxin-Ppr1 and alpha-elapitoxin-Pc1 were isolated from P. porphyriacus and P. colletti, respectively, using reverse-phase high performance liquid chromatography. Each toxin consisted of 62 amino acids with molecular weights of 6746.5Da and 6759.6Da, respectively. alpha-Elapitoxin-Ppr1 and alpha-elapitoxin-Pc1 caused concentration-dependent (0.1-0.3muM) inhibition of indirect twitches in the chick biventer cervicis nerve-muscle preparation. Both toxins inhibited contractile responses to exogenous ACh and CCh, but not KCl, suggesting a post-synaptic mode of action at the nicotinic acetylcholine receptor (nAChR). CCh concentration-response curves obtained in the presence or absence of alpha-elapitoxin-Ppr1 or alpha-elapitoxin-Pc1 indicated pA2 values of 6.97+/-0.03 and 7.04+/-0.07, respectively. Neither alpha-elapitoxin-Ppr1 (0.1muM) nor alpha-elapitoxin-Pc1 (0.1muM) had a significant effect on the electrically-induced twitches of the rat isolated phrenic nerve-diaphragm preparation. When the venom with the toxin removed (10mug/ml) was added to both the rat and chick preparations, the inhibition was significantly less than that caused by the intact whole venoms (10mug/ml). The current study shows that alpha-elapitoxin-Ppr1 and alpha-elapitoxin-Pc1 act as pseudo-irreversible antagonists at the nAChR of the skeletal neuromuscular junction and that the avian preparation is more sensitive to the neurotoxic effects of these toxins than the mammalian preparation.