The structural pathway of spontaneous microtubule nucleation was identified for the first time using recombinant mutant tubulin with a high nucleation rate, rapid flash-negative stain electron microscopy, and kinetic analyses of the size of critical nucleus. The single-stranded tubulin oligomers assembled during the early stage of polymerization had a variety of lengths and curvatures; however, the majority of these oligomers dissociated to tubulin. It was the few straight oligomers exceeding the critical size that underwent lateral interaction, had two-dimensional growth into sheets, and eventually closed into microtubules.