Transverse (t)-tubules are invaginations of the sarcolemma that facilitate excitation-contraction coupling in cardiac myocytes. T-tubules are absent or sparse at birth and develop postnatally, however this process is poorly understood, especially in the atria. Understanding how t-tubules develop normally and influence Ca2+ handling could inform strategies to restore t-tubules in disease. Here, we have used serial block-face scanning electron microscopy (SBFSEM) to reconstruct high-resolution 3-D models of t-tubule networks in sheep atrial myocytes from different developmental time points in order to understand how their morphology and density changes throughout postnatal development. Adult (~18 months; AD), 3 month (3M) and newborn (NB) sheep were euthanized using an overdose of pentobarbitone (200mg/kg) with heparin (10,000IU). Hearts were quickly excised and small portions (0.5mm cubes) were fixed in Karnovsky fixative then imaged using an FEI Quanta 250 FEG SEM with Gatan 3view System. Data are reported as means ± S.E.M using a one-way ANOVA and a Holm-Sadik post hoc test with n=5-9 cells. We found that some t-tubules were present at birth and t-tubule density (total t-tubule volume as % of cell volume) increased between NB and 3M myocytes (NB: 0.051 ± 0.029%; 3M: 0.6 ± 0.1%, p<0.001). There was no further increase in AD myocytes (0.41 ± 0.098%, p=0.10). We then set out to determine whether increased t-tubule density between NB and 3M was a result of increase in t-tubule size and/or number. There was an increase in t-tubules per unit volume from 4.1 ± 1.9 pL-1 (NB) to 27 ± 5.7 pL-1 (3M), (p<0.001), but no significant change in AD 22 ± 7.7 pL-1, p=0.378. Similarly, there was a concurrent increase in the average surface area of t-tubules (NB: 2.09 ± 0.56 µm2; 3M: 4.5 ± 0.63 µm2, p<0.05) with no difference between 3M and AD myocytes (AD: 2.82 ± 0.25 µm2, p=0.34). Unlike ventricular myocytes where t-tubules are relatively straight and uniform, we find that atrial t-tubules are morphologically diverse with branched, dilated or irregular phenotypes. Although present in adult, irregular t-tubules are more common at 3M. Our data indicated the presence of a higher proportion of very short ‘stump-like’ t-tubules in NB myocytes compared to AD cells (NB = 44%; AD = 23%) suggesting that atrial t-tubules may develop by invaginating from the sarcolemma to the cell centre. In summary, we have characterized the ultrastructure of atrial t-tubules during postnatal development for the first time. Our data suggest t-tubules develop over the first 3 months of life in the sheep due to both an increase in t-tubule number and size. Finally, although we observed no significant changes in density between 3M and AD atrial myocytes, it appears as though individual t-tubule morphology may still change and become less disordered with age, which warrants further investigation.