The fundamental principles of excitation-contraction coupling are highly conserved between vertebrate species. However, the role of one organelle in particular, the sarcoplasmic reticulum (SR), is highly variable among species, tissue-type, age and environmental conditions, and has been linked to heightened cardiac performance and numerous cardiac pathologies. The SR is a specialised form of endoplasmic reticulum found in muscle cells that provides an intracellular reservoir of calcium. SR calcium can be rapidly mobilised and released from the SR for contraction, and re-sequestered back into the SR for relaxation. In mammals, this intracellular calcium flux pathway is essential for normal cardiomyocyte excitation-contraction coupling. The role of the SR is more contentious in non-mammalian vertebrates with some species recruiting little or no calcium from the SR during excitation-contraction coupling. While most studies suggest athletic ectotherms rely more strongly on SR calcium cycling, phylogenetic investigations suggest other factors may be important, and environmental conditions are also major determinants. This talk will discuss the enigmatic role of the SR during the evolution of the four-chambered heart drawing on comparative studies across vertebrates.