The binding of postsynaptic neurotoxins from snake and marine cone snail ( Conus sp.) venoms to nicotinic acetylcholine receptor (AchR) was investigated with an ELISA-based, non-radioactive assay. Three snake postsynaptic toxins from the long-chain group ( Naja naja kaouthia cobratoxin, Naja oxiana neurotoxin I, Bungarus multicinctus α-bungarotoxin) and short-chain group ( Naja naja atra cobrotoxin, Naja oxiana neurotoxin II, and Laticauda semifasciata erabutoxin b) were studied. Both types of snake postsynaptic toxins showed a dose-response with constant AchR (50 μg/ml) and varying toxin concentrations (50-0.035 μg/ml). The minimum detection limits of the assay for snake toxins ranged from 310 to 1240 ng/ml (40–160 pmole/ml), depending on the toxin. Unlike any of the short-chain toxins, long-chain toxins consistently bound less receptor and reached maximum absorbance levels with toxin concentrations of 10–50 μg/ml. Competition for AchR binding between cone snail postsynaptic neurotoxins (conotoxins GI, MI, SI) and α-bungarotoxin or cobrotoxin resulted in a dose-response. The postsynaptic conotoxins were uniformly better competitors for AchR binding with α-bungarotoxin than with cobrotoxin. Heat stability studies with neurotoxin I, erabutoxin b, or cobrotoxin revealed a loss in AchR binding activity with increasing temperature. α-Bungarotoxin heated at 90°C had increased AchR binding activity by 105%, relative to 25°C samples, but lost the majority of its binding activity after 100°C. The enhanced binding of heated α-bungarotoxin to AchR was specific, as evidenced by a competitive dose-response with unheated α-bungarotoxin, but heated toxin lacked any biological activity in the mouse lethal assay. When conotoxins GI or MI were heated at 100°C, there was no detectable loss in AchR binding activity, and only a slight decrease in mouse lethality.