Transverse (t-) tubules are essential for facilitating the synchronous increase in cytosolic calcium during excitation-contraction coupling in mammalian ventricular myocytes. It has recently been established that atrial cells of large mammals, including humans, also possess substantial t-tubule networks1. Here we investigate the functional significance of t-tubules for synchronous Ca release in sheep atrial myocytes. Myocytes from adult sheep were enzymatically isolated from the left atrial appendage. Formamide was used to detubulate the cells2. Cells were bathed in Tyrode’s solution and stimulated via patch pipette. Confocal microscopy was used to obtain xy scans of a single z-plane of atrial myocytes using the Ca indicator Fluo-5F AM. Images were sampled at 50Hz and merged to produce pseudo-linescans. T-tests were used for statistical analysis. Pseudo-linescans showed triggered Ca release in control myocytes was synchronous, with a rapid rise at both sarcolemmal and central regions of the cell. In contrast, detubulated cells showed a rapid Ca rise at sarcolemmal sites and a slow Ca rise at central regions. To quantify the synchronicity of Ca release, rates of Ca rise for each region were plotted as a function of distance from the sarcolemma and fitted with a linear regression. The mean gradient was flat for control cells but was steep for detubulated cells (0.0015 ± 0.0085 vs. -0.075 ± 0.014, n=8-10; P<0.001) indicating a high degree of dyssynchrony. This resulted in a steep gradient for time to peak in detubulated cells (0.0006 ± 0.0014 vs. 0.0232 ± 0.0053, n=8-10; P<0.01). The difference in time to peak between central and sarcolemmal regions was significantly slower in detubulated than control cells (2.1 ± 14.5ms vs. 215.8 ± 39.5ms, n=8-9; P<0.001). Detubulated cells showed a marked latency in central Ca rise (6.4 ± 5.3ms vs. 68 ± 22.5ms, n=8-9; P<0.05). On exposure to isoprenaline (100nM) detubulated cell Ca transients from central regions showed a rapid Ca rise similar to those at the sarcolemmal regions. The steep gradient of dyssynchrony seen in detubulated cells was flattened in the presence of isoprenaline (-0.075 ± 0.014 vs. 0.009 ± 0.02, n=4; P<0.01). However, in spite of an increase in synchronicity of Ca release, the latency of Ca rise was still present (68 ± 22.5ms vs. 57.4 ± 8.6ms, n=4; P>0.05). These data show that Ca transients of sheep atrial myocytes result from synchronous Ca release at all regions of the cell. Acute loss of t-tubules resulted in dyssynchrony, increased latency and time to peak of Ca transients at central regions of the myocytes. β-adrenoceptor activation improves synchronicity of Ca release in detubulated myocytes but latency of Ca transients at the central regions persists due to the reliance of Ca wave propagation in the absence of t-tubules.