The current investigation aimed to develop a novel technique that allows the study of intracellular and intercellular Ca2+signalling in strips of atrial cardiomyocytes. There is evidence of cell heterogeneity within the atrial appendage, which may contribute to the failure in the treatment of antiarrhythias. Right atrial appendage biopsies from patients undergoing valve replacement surgery were placed in ice-cold Ca2+-free HEPES-buffered solution and transferred from the surgery to the laboratory on ice. Once in the laboratory, samples were placed in Ca2+-containing HEPES-buffered solution and 3 mm long strips of tissue were dissected from the appendage and loaded with the Ca2+-sensitive indicator Cal-520 AM (10µM) or Calbryte-520 AM (10µM). Real-time confocal microscopy was then utilized to observe fluctuations in Ca2+activity. The major advantage of this novel method is the minimal disturbance of cell-to-cell contacts within atrial bundles to allow the study of intercellular communication between atrial cardiomyocytes. We observed a different pattern of spontaneous Ca2+activity in atrial cardiomyocytes. Spontaneous activity varied from synchronized Ca2+spikes to asynchronous Ca2+waves and sparks. Another striking observation was the degree of atrial myocyte heterogeneity within muscle bundles. In conclusion, our preliminary investigation demonstrates how live confocal imaging of tissue strips isolated from the right atrial appendage is a useful technique for the study of intracellular and intercellular Ca2+signalling in the highly heterogeneous population of human atrial cardiomyocytes. A detailed study of the distinct atrial myocytes and their potential link to patient history may improve our understanding of atrial fibrillation and other arrhythmias, and inform how such diseases are treated in the future.