Atrial fibrillation (AF) commonly arises in the left atrial posterior wall (LAPW) and pulmonary veins (PVs). Dysregulated atrial sympathetic control is a central mechanism driving the induction and maintenance of AF. However, regional differences in atrial sympathetic structure and function remain poorly characterised. The present study used a fluorescence-based assay to evaluate sympathetic nerve distribution and single-terminal noradrenaline transporter (NAT) uptake kinetics throughout the rat atria. Furthermore, the impact of sympathetic stimulation on atrial electrical activity was assessed using a novel atrial optical mapping (OM) preparation with intact sympathetic chain innervation. Differences in distribution of sympathetic nerves were observed throughout the atria, with prominent clustering of large bundles in the LAPW and PVs leading to exaggerated overall spatial heterogeneity in these regions. Mean sympathetic single terminal NAT uptake rates were not different between regions, but there was high terminal-to-terminal variability. At baseline, OM revealed the LAPW as a site of significant conduction slowing, high action potential duration (APD) heterogeneity and a 'hot spot' for re-entrant arrhythmias. Stimulation of the left and right stellate ganglia induced tachycardia, a shift in site of first activation and enhanced atrial Ca2+ handling, but did not alter atrial conduction velocity (CV) or APD in any region. These data identify that the primary functional sequelae of adrenergic modulation in the healthy rat atria include positive chronotropy, altered activation patterns and modulation of Ca2+ dynamics, as distinct from APD and CV changes. Further exploration of atrial sympathetic nerve distribution and function in pathologies associated with increased AF risk is needed. KEY POINTS: Differences in sympathetic nerve distribution or function may underpin the predominance of atrial fibrillation development in the left atrial posterior wall (LAPW) and pulmonary veins. We demonstrate regional variation in the dispersion of sympathetic nerves throughout the healthy rat atria, with greatest heterogeneity observed in the LAPW and pulmonary veins. Sympathetic nerve single terminal noradrenaline transporter uptake rates were consistent in all atrial regions, but showed high terminal-to-terminal variability. Using a novel rat innervated atrial optical mapping preparation, we reveal that stimulation of the sympathetic chains elicits tachycardia, a shift in site of first activation, and enhances Ca2+ handling, but does not affect atrial conduction velocity or action potential duration. These data identify regional structural differences in sympathetic nerves in the healthy rat atria and demonstrate that the primary functional sequelae of adrenergic modulation are positive chronotropy, changes in activation patterns and modulation of Ca2+ dynamics.