Preparations of calcium-tolerant cardiac myocytes for studies on intracellular calcium ([Ca2+ ]i ) signaling and contraction have been difficult owing to the susceptibility of these cells to the enzymatic digestion process. This often leads to the cells acquiring a bricklike contracted shape with a granular cytoplasm and membrane blebbing. A successful preparation results in single myocytes that do not show spontaneous contractions and that remain relaxed in Ca2+ -containing buffers, indicating that the sarcolemmal membrane was not damaged during enzymatic digestion and led to membrane depolarization.