Huwentoxin-I (HWTX-I) is a neurotoxic peptide purified from the venom of the Chinese bird spider Selenocosmia huwena. The effects of HWTX-I on neuromuscular transmission of vertebrate skeletal muscle have been investigated by means of twitch tension and electrophysiological techniques. On isolated mouse phrenic nerve-hemidiaphragm preparations, HWTX-I blocked the twitch responses to indirect, but not to direct, muscle stimulation. The time needed for complete block of the neuromuscular transmission was dose dependent. The transmission could be mostly restored by prolonged repeated washing with Tyrode's solution. If the preparation was pretreated with D-tubocurarine and then immersed in a mixed solution of D-tubocurarine and HWTX-I, the washout time necessary to restore the neuromuscular transmission was significantly decreased. Intracellular recording at the end-plate region of frog sartorius muscle revealed that HWTX-I could synchronously reduce the amplitude of the acetylcholine potential induced by ionophoretic application of acetylcholine as well as the amplitude of the end-plate potential evoked by nerve stimulation. Both of these effects eventually disappeared; however, both could be restored by prolonged washing. Experiments on Xenopus embryonic myocytes indicated that HWTX-I reduced the open probability of acetylcholine-induced channel activity, and finally blocked the channel. All of these results demonstrated that HWTX-I was a peptide neurotoxin and the postsynaptic nicotinic acetylcholine receptor was its site of action.