It was shown that the temperature sensitivity of shortening velocity of skeletal muscles is higher at temperatures below physiologic (10-25°C) than at temperatures closer to physiologic (25-35°C) and is higher in slow than fast muscles. However, as intact muscles invariably express several myosin isoforms they are not the ideal model to compare the temperature sensitivity of slow and fast myosin isoforms. Moreover, temperature sensitivity of intact muscles and single muscle fibres cannot be unequivocally attributed to a modulation of myosin function itself as in such specimen myosin works in the structure of the sarcomere together with other myofibrillar proteins. We have used an in vitro motility assay (IVMA) approach in which the impact of temperature on velocity can be studied at a molecular level as in such assay acto-myosin interaction occurs in the absence of sarcomere structure and of the other myofibrillar proteins. Moreover, the temperature modulation of velocity could be studied in pure myosin isoforms (rat type 1, 2A and 2B and rabbit type 1 and 2X) that could be extracted from single fibers and in a wide range of temperatures (10-35°C) as isolated myosin is stable up to physiologic temperature. The data show that at molecular level, the temperature sensitiveness is higher at lower (10-25°C) than at higher (25-35°C) temperatures, consistent with experiments on isolated muscles. However, slow myosin isoforms did not show a higher temperature sensitivity than fast isoforms on the contrary of what observed in intact slow and fast muscles