This method is a modification of the initial procedure employed to purify tubulin from mammalian brain. It consists of tissue homogenization, elimination of cell membranes, ammonium sulfate fractionation, and batch anion exchange, followed by selective precipitation with magnesium chloride. Half gram of electrophoretically homogenous, active, concentrated calf brain tubulin is typically purified in 9 h, dialyzed overnight, and stored under liquid nitrogen. Prior to use the protein is equilibrated in the experimental buffer and its concentration measured. This tubulin preparation has been very extensively characterized. Frozen aliquots have been found to retain microtubule assembly activity after 10 yr of storage.