The κ-neurotoxins are useful ligands for the pharmacological characterization of nicotinic acetylcholine receptors because they are potent antagonists at only a subgroup of these receptors containing either α3- or α4-subunits (IC50 ≤ 100 nM). Four of these highly homologous, 66 amino acid peptides have been purified from the venom of Bungarus multicinctus [κ-bungarotoxin (κ-Bgt), κ2-Bgt, κ3-Bgt] and Bungarus flaviceps [κ-flavitoxin (κ-Fvt)]. Two approaches were taken to examine the binding of these toxins to nicotinic receptors. First, venom-derived κ-Fvt and κ-Bgt were radioiodinated and the specific binding was measured of these toxins to overlapping synthetic peptides (16-20 amino acids in length) prepared based on the known sequence of the nicotinic receptor α3-subunit. At least two main regions of interaction between the toxins and the receptor subunit were identified, both lying in the N-terminal region of the subunit that is exposed to the extracellular space. The second approach examined the importance of several sequence positions in κ-Bgt for binding to α3-containing receptors in autonomic ganglia and cell-containing muscle receptors, This was done using site-directed mutants of κ-Bgt produced by an Escherichia coli expression system. Arg-34 and position 36 were important for binding to both receptor subtypes, while replacing Gln-26 with Trp-26 (an invariant in α-neurotoxins) increased affinity for the muscle receptor by 8-fold. The results confirm that κ-neurotoxins bind potently to the α3-subunit and bind with considerably reduced affinity (K(d) ~ 10 μM) to muscle receptors. Site-directed mutagenesis of recombinant κ-Bgt is thus an important approach for the study of structure-function relationships between κ-Bgt and nicotinic receptors.