The rapid cooling (RC) response in muscle is an increase in cytoplasmic [Ca²⁺] that is probably caused by Ca²⁺ release from the sarcoplasmic reticulum (SR). However, the molecular bases of this response have not been completely elucidated. Three different isoforms of the SR Ca²⁺ release channels, or ryanodine receptors (RyRs), have been isolated (RyR1, RyR2, and RyR3). In the current investigation, the RC response was studied in RyR-null muscle cells (1B5) before and after transduction with HSV-1 virions containing the cDNAs encoding for either RyR1, 2, or 3. Cells were loaded with Fluo4-AM to monitor changes in [Ca²⁺]_i and perfused with either cold (~0°C), room temperature (RT), or RT buffer containing 40mM Caffeine. Control cells showed no significant response to cold or to caffeine, while robust Ca²⁺ transients were recorded in response to both rapid cooling and caffeine in transduced cells expressing any one of the three RyR isoforms. Our data demonstrate directly that RyRs are responsible for the RC response, and that all three isoforms respond in a similar manner. Ca²⁺ release from RyRs is likely caused by a rapid cooling-induced conformational change of the channel from the closed to the open state.