Steps involved in excitation-contraction coupling in mammalian myocardium have been derived using a relatively limited number of animal species. However, the use of animal models for investigations into excitation-contraction coupling in normal and disease states has encompassed a wide range of animal species. We addressed the question as to whether excitation-contraction coupling as currently understood applies to intracellular calcium handling in myocardium from multiple mammalian species, amphibian, and avian myocardium. The bioluminescent calcium indicator aequorin was used to record intracellular calcium transients in both ventricular and atrial tissue. We report that in all mammalian and avian species studied the calcium transient recorded in both ventricular and atrial myocardium is monophasic and reflects calcium release and re-uptake by the sarcoplasmic reticulum. In contrast, the Ca2+ transient recorded from salamander myocardium is prolonged relative to mammalian and avian myocardium, and appears to reflect in part trans-sarcolemmal calcium entry. Only in diseased myocardium derived from human and swine myocardium was a second component detected in the calcium transient. These data indicate that sarcoplasmic reticulum calcium handling is pivotal in excitation-contraction coupling for multiple species with differing physiologies. Also, in disease states, intracellular calcium handling is often affected with resultant alterations in the time-course and/or configuration of the calcium transient.