Objective: The parasympathetic (P) nervous system is thought to contribute significantly to focal atrial fibrillation (AF). Thus, we hypothesized that P nerve fibers (and related muscarinic receptors) are preferentially located in the posterior left atrium (PLA) and that selective cholinergic blockade in the PLA can be successfully performed to alter vagal AF substrate. Methods: PLA, pulmonary veins (PVs) and left atrial appendage (LAA) from 6 dogs were immunostained for sympathetic (S) nerves, P nerves, and muscarinic (M2) receptors. Epicardial electrophysiologic mapping was performed in 7 additional dogs. Results: The PLA was the most richly innervated with nerve bundles containing P and S fibers (PV=0.9±1, PLA=3.2±2.5, LAA=0.17±0.3/cm²; p<0.001); nerve bundles were located in fibrofatty tissue as well as in surrounding myocardium. P predominated over S fibers within bundles (P/S: PV=4.4, PLA=7.2, LAA=5.8). M2 distribution was also most pronounced in the PLA (M2 stained cells/cm²: PLA=17.8±8.3, PV=14.3±7.3, LAA=14.5±8, p=0.012). Left cervical vagal stimulation (VS) caused significant ERP shortening in all regions, with easily inducible AF. 1% tropicaimide was then applied topically to the PLA. Following tropicaimide application, VS-induced ERP shortening was significantly attenuated in the PLA, PV and LAA; AF induciblity decreased by 92% (p<0.001). Conclusions: 1) P fibers and M2 receptors are preferentially located in the PLA, suggesting an important role for this region in creation of vagal AF substrate and 2) targeted P blockade in the PLA is feasible and results in an attenuation of vagal responses in the entire left atrium, with a consequent change in AF substrate.