Allelic variation in CHRNA3 , the gene encoding the α3 nicotinic acetylcholine receptor (nAChR) subunit, increases vulnerability to tobacco dependence and smoking-related diseases, but little is known about the role for α3-containing (α3*) nAChRs in regulating the addiction-related behavioral or physiological actions of nicotine. α3* nAChRs are densely expressed by medial habenula (mHb) neurons, which project almost exclusively to the interpeduncular nucleus (IPn) and are known to regulate nicotine avoidance behaviors. We found that Chrna3tm1.1Hwrt hypomorphic mice, which express constitutively low levels of α3* nAChRs, self-administer greater quantities of nicotine (0.4 mg kg−1 per infusion) than their wild-type littermates. Micro-infusion of a lentivirus vector to express a short-hairpin RNA into the mHb or IPn to knock down Chrna3 transcripts markedly increased nicotine self-administration behavior in rats (0.01-0.18 mg kg−1 per infusion). Using whole-cell recordings, we found that the α3β4* nAChR-selec...