Introduction: Important differences in Ca2+ signaling occurred between ventricular and atrial cardiomyocytes has been attributed to the lack of a regular T-tubular system and distinct distribution of atrial L-type Ca2+ channels (LTCCs). In addition to critical subpopulation of LTCCs localized to dyadic junctions, extradyadic channels associated with district regions of surface membrane have been distinguished. Hypothesis: Here, we hypothesise that a subpopulation of LTCCs housed in caveolae microdomains, could play an important role in modulation of Ca2+ signaling, particularly in cells lacking T-tubules such as atrial cardiomyocytes. Methods: Scanning ion conductance, confocal, and electron microscopy were used to characterize membrane topography, caveolae and T-tubular network in adult rat atrial cardiomyocytes. Ca2+-sensitive fluorescent dye Fluo-4AM was used to monitor changes in [Ca2+]i. Super-resolution scanning patch-clamp was applied to identify distribution of functional LTCCs, before and after caveolae depletion by methyl-β-cyclodextrin (MβCD). Results: MβCD abolished ~60% caveolae and significantly decreased occurrence of spontaneous calcium events (from 1.64±0.22 events/cell at baseline to 0.57±0.15 events/cell after MβCD treatment, P<0.001). At baseline, functional atrial LTCCs were found both in the T-tubules and in the crest areas of the sarcolemma with similar occurrence. While MβCD did not affect LTCCs occurrence in the T-tubules (29% vs. 33%, before and after MβCD treatment, NS), it completely abolished occurrence of LTCCs on the crest of sarcolemma (33% vs. 0% before and after MβCD treatment, P<0.001). No changes in cell topography and T-tubule openings were observed after MβCD treatment. Along with changes in LTCCs distribution, MβCD decreased response to both β1- (6.52±1.42 events/cell vs. 2.66±0.59 events/cell before and after MβCD, P<0.05) and β2-ARs stimulation (3.20±0.58 events/cell vs. 1.45±0.21 events/cell before and after MβCD, P<0.05). Conclusions: Our results provide the first direct evidence of caveolae specific localization and regulation of functional LTCCs in atrial cardiomyocytes and suggest their possible role in the mechanism of unique atrial calcium cycling.