1. The effects of three scorpion toxins, charybdotoxin (CTX), iberiotoxin (IbTX), and noxiustoxin (NTX) have been studied on acetylcholine release and on K+ channels by means of twitch tension and electrophysiological recording techniques using isolated skeletal muscle preparations and by a radioligand binding assay using 125I-labelled dendrotoxin I (DpI) and rat brain synaptosomal membranes. 2. On chick biventer cervicis preparations, CTX and IbTX (125 nM) augmented the twitch responses to indirect muscle stimulation. Further, the increase (about 70-80% of control twitch height) was fast in onset, reaching a maximum within 25-30 min. NTX at 125 nM produced a slower augmentation of the twitch responses to indirect muscle stimulation, with the maximum response being seen after 40-50 min. 3. On mouse triangularis sterni preparations, CTX (300 nM after 35-40 min) and IbTX (100 nM after 15 min) increased quantal content of the evoked endplate potentials (e.p.p.) by about two fold. However, NTX (300 nM) caused only a small increase in e.p.p. amplitude, which was followed by repetitive e.p.ps in response to single shock nerve stimulation after 40-50 min. 4. Extracellular recording of nerve terminal current waveforms in triangularis sterni preparations revealed that CTX and IbTX (3-100 nM), but not NTX (100 nM), blocked the Ca(2+)-activated K+ current, IK-Ca. However, there was no major change in the portion of the nerve terminal waveform associated with voltage-dependent K+ currents, IKv. 5. In the radioligand binding assay, NTX potently displaced labelled [125I]-DpI, whereas CTX produced only partial displacement. However, IbTX did not displace [125I]-DpI from its binding sites on rat brain synaptosomal membranes.6. We conclude that these three structurally homologous scorpion toxins act on different K+ channels and that this leads to different patterns of facilitation of acetylcholine release. IbTX acts selectively on high conductance Ca2+-activated K+ channels, leading to an increase in the amplitude of e.p.ps without any other changes. NTX acts on voltage-dependent K+ channels that are sensitive to dendrotoxin and causes repetitive e.p.ps. CTX shares amino acid residues that exist in the structures of IbTX and NTX;CTX acts on both Ca2+- and voltage-dependent K+ channels.