Episodes of atrial fibrillation (AF) in patients are more prevalent at night, and cardiomyocytes in the pulmonary vein (PV) sleeves are a major source of ectopic activity driving AF.  While it is known that circadian clocks within heart muscle cells contribute to the control of pacemaking and ventricular repolarisation, the mechanisms underlying the nighttime preponderance in AF are unknown. We aimed to address the hypothesis that circadian clocks exist within PV cardiomyocytes, controlling their electrophysiology and susceptibility to pro-arrhythmic activity.  Animal procedures were approved by the University of Bristol Animal Welfare and Ethics Review Board and conducted in accordance with UK law. Male Wistar rats were maintained in a 24-hour cycle of 12-hr light/dark (lights-on at Zeitgeber time, ZT=0; lights-off, ZT12) and hearts were removed at ZT=0, 6, 12 or 18 hr under terminal general anaesthesia (140 mg/kg Na pentobarbital i.p.). RNA was extracted from left atrial (LA) appendage (LAA) and proximal PV at the LA/PV junction (3 rats per ZT) and RNA sequencing conducted. Reads were mapped to the rat genome, counts normalised and models in which ZT was or was not included as a factor compared (Likelihood Ratio Test, adjusted-P<0.01). Whole-cell current clamp recordings were made from cardiomyocytes isolated from the proximal PV (n=308) and the LAA (n=264) (N=74 rats). The effects of noradrenaline (NA, 1 µM) and acetylcholine (ACh, 1 µM) were examined. Data were plotted against the ZT of the time of recording and fitted to a sine wave to establish circadian rhythmicity (P<0.05, extra-sum-of-squares F-test). The effect of ≥24 hr constant dark in the period immediately before experiment was examined. Data are reported as mean ± standard error.  The expression of 1368 genes varied significantly with ZT, including circadian clock components (e.g. Bmal1).  PV cells were larger than LAA cells (73±1.6 pF vs 53±1.3 pF, P<0.0001) and had more depolarised resting membrane potential (-69±0.2 mV vs -72±0.1 mV, P<0.0001). Both cell types showed circadian variation in action potential duration at 90% repolarisation (APD₉₀) and frequency of pro-arrhythmic activity. Pro-arrhythmic activity was greatest in PV cells and the frequency was greater during the rest phase (ZT0-12) in both cell types. In contrast, the circadian rhythm in APD₉₀ differed between cell type, with the longest APD­­₉₀ recorded during the resting phase in PV cells (ZT12-24) but during the active phase in LAA cells. Pro-arrhythmic activity was increased by NA and decreased by ACh in both cell types, with the maximal effect of either neurotransmitter during the rest phase.  Circadian variation in APD₉₀ and proarrhythmic-activity has been demonstrated in isolated proximal PV and LAA cardiomyocytes, with differences in rhythm between the two cell types. RNA sequencing suggests the presence of peripheral clocks in LAA and PV cardiomyocytes. Understanding circadian rhythms in the pro-arrhythmic activity of PV cardiomyocytes is likely to be valuable in the development of future therapeutic options for AF.