The subunit structure of rat brain synaptosomal tubulin was examined by high resolution two-dimensional gel fractionation. Whole brain cytoplasmic tubulin consists of two groups of alpha subunits (alpha1 and alpha2), and a minimum of two beta subunits (beta1 and beta2). Both alpha subunits consist of a major relatively acidic form and minor relatively basic forms. In contrast, tubulin purified from synaptoplasm contains an additional subunit, alpha3, which has the same isoelectric point but slightly faster electrophoretic mobility than alpha1 and alpha2. All synaptosomal alpha subunits are the relatively acidic forms and the minor basic forms are absent. The synaptosomal beta subunits have electrophoretic properties similar to the corresponding cytoplasmic forms. The alpha3 synaptosomal tubulin subunit has affinity for colchicine, has a tryptic peptide map similar to whole brain cytoplasmic alpha tubulin, and can be purified by a standard tubulin purification method.