We have developed methods using video‐enhanced differential interference contrast light microscopy (VE‐DIC) to measure the association and dissociation rate constants and transition frequencies of microtubule dynamic instability for microtubules assembled from pure tubulin, plus brain microtubule assoicated proteins (MAPs), and for microtubule assembly in living cells and cytosol extracts. Following nucleation, a microtubule end is seen to elongate at constant velocity until it abruptly begins rapid shortening, a transition termed catastrophe. The microtubule either disappears, or converts back to the elongation phase, a transition termed rescue. Catastrophes and rescues occur stochastically and infrequently in comparison to the durations of the elongation and shortening phases. In purified tubulin preparations from both mammalian brain and sea urchin embryos, the elongation and shortening phases exhibit distinctly different association and dissociation rate constants; in particular, the rate of dissocation during rapid shortening can be 100 times or more greater than during elongation particularly at high Mg2+.Brain MAPs (MAP2and Tau) promote faster elongation, but suppress dynamic instability mainly by decreasing the frequency of catastrophe and increasing the frequency of rescue. In contrast, there are unknown factors in living dividing cells and in extracts from dividing cells which enhance dynamic instability by producing high frequencies of catastrophe (.01–.05 sec−1) at fast elongation velocities (10 &mgr;m min−1). Using a microscope perfusion chamber, we have shown for microtubules assembled from pure tubulin that dilution induces rapid shortening within several seconds independent of the elongation velocity or microtubule length. Thus, the stabilizing cap at elongating microtubule ends is small and sensitive to transient changes in the rate of tubulin association, even at high elongation velocities. This means that substantial changes in microtubule assembly can be regulated by cellular factors which increase the frequency of catastrophe by transiently interfering with tubulin association at elongating microtubule ends without significantly effecting the average rate of elongation.