We report here the construction of a novel knock-in mouse expressing chimeric α3 nicotinic acetylcholine receptor (nAChR) subunits with pharmacological sensitivity to α-bungarotoxin (αBTX). Sensitivity was generated by substituting five amino acids in the loop C (β9–β10) region of the mouse α3 subunit with the corresponding residues from the α1 subunit of the muscle type receptor from Torpedo californica. To demonstrate the utility of the underlying concept, expressed α3 subunits were characterized in the superior cervical ganglia (SCG) of homozygous knock-in mice, where the synaptic architecture of postsynaptic α3-containing nAChR clusters could now, for the first time, be directly visualized and interrogated by live-staining with rhodamine-conjugated αBTX. Consistent with the postsynaptic localization of ganglionic nAChRs, the αBTX-labeled puncta colocalized with a marker for synaptic varicosities. Following in vivo deafferentation, these puncta persisted but with significant changes in intensity and distribution that varied with the length of the recovery period. Compound action potentials and excitatory postsynaptic potentials recorded from SCG of mice homozygous for α3 were abolished by 100 nmαBTX, even in an α7 null background, demonstrating that synaptic throughput in the SCG is completely dependent on the α3-subunit. In addition, we observed that the genetic background of various inbred and outbred mouse lines greatly affects the functional expression of α3-nAChRs, suggesting a powerful new approach for exploring the molecular mechanisms underlying receptor assembly and trafficking. As αBTX-sensitive sequences can be readily introduced into other nicotinic receptor subunits normally insensitive to αBTX, the findings described here should be applicable to many other receptors.