Abstract DeCuevas et al. [J. Cell Biol. 116 (1992) 957–965] demonstrated by circular dichroism spectroscopy for the kinesin stalk fragment that shifting temperature from 25 to 30°C caused a conformational transition. To gain insight into functional consequences of such a transition, we studied the temperature dependence of a full-length kinesin by measuring both the velocity of microtubule gliding across kinesin-coated surfaces and microtubule-promoted kinesin ATPase activity in solution. The corresponding Arrhenius plots revealed distinct breaks at 27°C, corroborating the temperature-dependent conformational transition for a motility-competent full-length kinesin. Microtubules were found to glide up to 45°C; at higher temperatures, kinesin was irreversibly damaged.