Microtubule assembly is enhanced by the addition of 1 M sucrose or 4 M glycerol to the reassembly mixture. Tubulin can be purified from guinea pig brain readily and in good yield by two cycles of assembly in glycerol-containing solutions. The tubules assembled in glycerol and sucrose are more stable than tubules formed in the absence of these compounds. Assembly occurs in glycerol or sucrose in the absence of ATP or GTP, but is greatly accelerated by their presence. Cytoplasmic microtubules in vivo are remarkable for their ability to rapidly assemble and disassemble in response to a wide variety of chemical agents and physical conditions. Recently, Weisenberg demonstrated that the addition of chelating agents such as EDTA or EGTA and GTP or ATP to the supernatants of centrifuged brain homogenates allows the reassembly of microtubules in vitro. The reassembly proceeds rapidly at 37°C, is reversed by lowering the temperature to 4°C, and is inhibited by colchicine and by calcium. While it is likely that calcium is an important factor in the physiological control of microtubule assembly, the role of the nucleotide triphosphate is also of interest. The microtubular protein, tubulin, has 1 mol of tightly bound guanine nucleotide per 6S dimer if GTP is present in the medium. The nonexchangeable, tightly bound, nucleotide is mostly in the diphosphate form. On incubation with GTP, the γ-phosphate of the GTP that is added is hydrolyzed and transferred to the GDP at the nonexchangeable site, converting it to GTP. The role of this transphosphorylation in the assembly of microtubules is unclear, since attempts to study the fate of GTP during assembly have been hampered by the lability of the tubules formed. To circumvent this problem, we have used various conditions that favor polymerization, and have found that polymerization of tubules may occur without the addition of nucleotide. Tubulin can be purified on the basis of its polymerization and depolymerization. The results demonstrated that tubulin could be purified on the basis of its polymerization and depolymerization. The tubules assembled without GTP are of normal morphology. However, in the samples assembled in 4 M glycerol and GTP, numerous abnormal assembly forms are seen in addition to normal tubules. These forms are composed of protofilaments identical in appearance to those of the microtubule, but appear to be opened in a sheet rather than rolled into a tubule. The stability of the tubules formed in glycerol and sucrose differs from those formed in reassembly buffer. The tubules in glycerol showed no depolymerization at 4°C or at −Microtubule assembly is enhanced by the addition of 1 M sucrose or 4 M glycerol to the reassembly mixture. Tubulin can be purified from guinea pig brain readily and in good yield by two cycles of assembly in glycerol-containing solutions. The tubules assembled in glycerol and sucrose are more stable than tubules formed in the absence of these compounds. Assembly occurs in glycerol or sucrose in the absence of ATP or GTP, but is greatly accelerated by their presence. Cytoplasmic microtubules in vivo are remarkable for their ability to rapidly assemble and disassemble in response to a wide variety of chemical agents and physical conditions. Recently, Weisenberg demonstrated that the addition of chelating agents such as EDTA or EGTA and GTP or ATP to the supernatants of centrifuged brain homogenates allows the reassembly of microtubules in vitro. The reassembly proceeds rapidly at 37°C, is reversed by lowering the temperature to 4°C, and is inhibited by colchicine and by calcium. While it is likely that calcium is an important factor in the physiological control of microtubule assembly, the role of the nucleotide triphosphate is also of interest. The microtubular protein, tubulin, has 1 mol of tightly bound guanine nucleotide per 6S dimer if GTP is present in the medium. The nonexchangeable, tightly bound, nucleotide is mostly in the diphosphate form. On incubation with GTP, the γ-phosphate of the GTP that is added is hydrolyzed and transferred to the GDP at the nonexchangeable site, converting it to GTP. The role of this transphosphorylation in the assembly of microtubules is unclear, since attempts to study the fate of GTP during assembly have been hampered by the lability of the tubules formed. To circumvent this problem, we have used various conditions that favor polymerization, and have found that polymerization of tubules may occur without the addition of nucleotide. Tubulin can be purified on the basis of its polymerization and depolymerization. The results demonstrated that tubulin could be purified on the basis of its polymerization and depolymerization. The tubules assembled without GTP are of normal morphology. However, in the samples assembled in 4 M glycerol and GTP, numerous abnormal assembly forms are seen in addition to normal tubules. These forms are composed of protofilaments identical in appearance to those of the microtubule, but appear to be opened in a sheet rather than rolled into a tubule. The stability of the tubules formed in glycerol and sucrose differs from those formed in reassembly buffer. The tubules in glycerol showed no depolymerization at 4°C or at −