Strongylocentrotus purpuratus outer doublet microtubules were prepared by extraction of sperm tail axonemes with 0.6 m-KCl. Sonication of the outer doublet microtubules in 5 m m-2-( N-morpholino)ethanesulphonic acid, 1 m m-ethyleneglycol-bis-(β-aminoethyl ether) N,N′-tetraacetic acid, 1 in m-MgSO 4 (pH 6.7) solubilized up to 35% of the outer doublet protein, depending on the power input, in a manner which was non-selective for either subfiber. Tubulin comprised 75 to 85% of the total solubilized protein in a 200,000 g supernatant obtained from the sonicated suspension. Colchicine-binding assays demonstrated that the tubulin was largely in a native form ( K A = 10 6, liters mole −; 0.74 mole of colchicine bound per mole of tubulin at infinite concentration of colchicine). Microtubule self-assembly from the 200,000 g supernatants in the absence of added seeds or glycerol was quantitated by light-scattering at 350 nm. The critical protein concentration for assembly was 0.55 mg ml −1 at 37 °C and the reaction occurred optimally in the presence of 2 m m-GTP and 150 m m-KCl. The solubilized outer doublet tubulin formed singlet microtubules upon reassembly under our in vitro conditions. The authenticity of the microtubules was verified by both negative stain and thin-section electron microscopy. Polymerization was prevented by colchicine and podophyllotoxin, and depolymerization occurred rapidly on cooling the microtubules to 0 °C. The susceptibility of the reassembled microtubules to low temperature suggested that they could be “recycled” by the warm assembly-cold disassembly procedure developed for vertebrate brain (Borisy et al., 1974). Twice recycled outer doublet tubulin was devoid of high molecular weight microtubule-associated proteins, as judged by gel electrophoresis in the presence of sodium dodecyl sulfate. However, trace amounts (less than 5%) of intermediate molecular weight material was visible on heavily overloaded gels. The function of this material is uncertain, but it is not chemically equivalent to the tau factor of vertebrate brain (Weingarten et al., 1975), since it cannot be separated from the tubulin by phosphocellulose adsorption. In addition, phosphocellulose-treated tubulin reassembled to the same extent as untreated tubulin, suggesting that the reassembly of outer doublet tubulin does not require the protein equivalents of brain microtubule-associated proteins or tau factor. If accessory proteins are required for the reassembly of outer doublet tubulin, they are not removed by phosphocellulose under the conditions employed, and they must comprise less than 5% of the total protein.