We report here the construction of a novel knock-in mouse expressing chimeric alpha3 nicotinic acetylcholine receptor (nAChR) subunits with pharmacological sensitivity to alpha-bungarotoxin (alphaBTX). Sensitivity was generated by substituting five amino acids in the loop C (beta9-beta10) region of the mouse alpha3 subunit with the corresponding residues from the alpha1 subunit of the muscle type receptor from Torpedo californica. To demonstrate the utility of the underlying concept, expressed alpha3[5] subunits were characterized in the superior cervical ganglia (SCG) of homozygous knock-in mice, where the synaptic architecture of postsynaptic alpha3-containing nAChR clusters could now, for the first time, be directly visualized and interrogated by live-staining with rhodamine-conjugated alphaBTX. Consistent with the postsynaptic localization of ganglionic nAChRs, the alphaBTX-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 alpha3[5] were abolished by 100 nmalphaBTX, even in an alpha7 null background, demonstrating that synaptic throughput in the SCG is completely dependent on the alpha3-subunit. In addition, we observed that the genetic background of various inbred and outbred mouse lines greatly affects the functional expression of alpha3[5]-nAChRs, suggesting a powerful new approach for exploring the molecular mechanisms underlying receptor assembly and trafficking. As alphaBTX-sensitive sequences can be readily introduced into other nicotinic receptor subunits normally insensitive to alphaBTX, the findings described here should be applicable to many other receptors.