A series of novel, potent neuronal nicotinic acetylcholine receptor (nAChR) ligands derived from 3,6-diazabicyclo[3.2.0]heptane have been synthesized and evaluated for binding affinity and agonist activity at the alpha4beta2 nAChR subtype. Structure-activity relationship studies of these novel nAChR ligands focused on substitution effects on the pyridine ring, as well as stereo- and regiochemical influences of the 3,6-diazabicyclo[3.2.0]heptane core. Small 5-substituents on the pyridine ring had a modest impact on the binding affinities and functional activities. 6-Bromo, 6-chloro, and 6-methyl substituents on the pyridine ring led to increased binding affinities and improved functional activities. Most of the 6-N-pyridinyl-substituted 3,6-diazabicyclo[3.2.0]heptanes are selective for the alpha4beta2 nAChR subtype. Compounds (1R,5S)-25, (1R,5S)-55, and (1R,5S)-56 were virtually inactive as agonists at the halpha3beta4 nAChR but retained potency and efficacy at the halpha4beta2 nAChR subtype. 3-N-Pyridinyl-substituted series demonstrated more complex SAR. (1R,5R)-39, (1R,5R)-41, and (1R,5R)-42 were found to be much more potent at the halpha3beta4 nAChR subtype, whereas (1R,5R)-38 and (1R,5R)-40 were very selective at the halpha4beta2 nAChR subtype. The SAR studies of these novel ligands led to the discovery of several compounds with interesting in vitro pharmacological profiles.