Microtubules are dynamically unstable polymers that interconvert stochastically between polymerization and depolymerization. Compared with microtubules assembled from purified tubulin, microtubules in a physiological environment polymerize faster and transit more frequently between polymerization and depolymerization. These dynamic properties are essential for the functions of the microtubule cytoskeleton during diverse cellular processes. Here, we have reconstituted the essential features of physiological microtubule dynamics by mixing three purified components: tubulin; a microtubule-stabilizing protein, XMAP215; and a microtubule-destabilizing kinesin, XKCM1. This represents an essential first step in the reconstitution of complex microtubule dynamics-dependent processes, such as chromosome segregation, from purified components.