The ryanodine receptor ion channels (RyRs) release Ca2+ from the endo/sarcoplasmic reticulum in a variety of nonvertebrate and vertebrate species including flies, crustaceans, birds, fish, and amphibians. They are most abundant in skeletal and cardiac muscle, where in response to an action potential, the release of Ca2+ ions from the sarcoplasmic reticulum through the RyRs into the cytoplasm leads to muscle contraction (i.e., excitation-contraction coupling). Here, we describe how to determine their cellular location using isoform-specific antibodies, their protein levels using an in vitro (3 H)ryanodine-binding assay, and their cellular release of Ca2+ using RyR-specific channel agonists and inhibitors.