Atrial fibrillation (AF) is the most common form of sustained cardiac arrhythmia, with age being a significant risk factor. Inside the pulmonary veins a sheath of cardiomyocytes called pulmonary vein sleeve cells (PVCs) can be found. PVCs can cause atrial fibrillation by generating spontaneous increases in the intracellular Ca2+ concentration and ectopic electrical activity. Ultrastructural changes during atrial fibrillation have been described in atrial cardiomyocytes. Besides an increase in the number of glycogen granules, the reported changes include a greater variability in the number and size of mitochondria. However, the published data are inconsistent; some studies reported an increase of mitochondrial number and size in AF models, whereas other studies reported a decrease. So far, no studies have been performed to investigate changes in the mitochondrial structure occurring in PVCs. Since age is a significant risk factor for the development of AF we expected that the ultrastructure and/or number of mitochondria in PVCs would change with age. Here, we describe the results of a comparative ultrastructural study of pulmonary vein sleeve cells, atrial and ventricular cardiomyocytes from 3 and 24 month-old mice. PVCs were isolated from mouse lung slices after agarose-inflation of the lungs. Atrial and ventricular cardiomyocytes were prepared from wedge-shaped sections of mouse hearts. We counted the number, and measured the size, of mitochondria in 25 µm2 sections of three to five EM images in four animals per age group. The quantification showed an increased number of mitochondria in PVCs from the older animals (14.2 ± 0.9 vs. 19.6 ± 1.2 mitochondria / 25 µm2, 3 vs. 24 month respectively, P < 0.05, unpaired t-test, n = 4 animals per group). Additionally, mitochondria were significantly enlarged (0.7 ± 0.02 vs. 1.0 ± 0.1 µm2, 3 vs. 24 month respectively, P < 0.0001, unpaired t-test, n = 4 animals per group). Values are means ± S.E.M. In contrast, we did not see any significant differences in mitochondrial number or size in atrial and ventricular cardiomyocytes. Besides the changes in mitochondria, we found the presence of lipofuscin in PVCs, atrial and ventricular myocytes from the older animals. Lipofuscin is a known marker for ageing, and contains larger amounts of oxidated unsaturated fatty acids and non-degraded non-functional mitochondria. These results show age associated structural changes in pulmonary vein sleeve cells. Since mitochondria are an important Ca2+ buffer in cardiomyocytes, an increase in mitochondrial number and size could contribute to the pro-arrhythmic signalling described for pulmonary vein sleeve cells.