Tubulin from either outer doublet microtubules or the central pair of microtubules in the starfish sperm flagellum was resolved into two alpha-subunits and one beta-subunit by two-dimensional gel electrophoresis. Further fractionation of the doublet microtubules into B-tubules, A-tubules, and 'partitions,' i.e. a part of the A-tubule wall shared with the B-tubule, revealed that all these fractions contained two alpha-subunits (alpha 1 and alpha 2) and one beta-subunit of tubulin, the ratios of alpha/beta being almost one. Both the B-tubule and the wall of the A-tubule exclusive of the partition consisted of alpha 1- and alpha 2-subunits in addition to beta-subunits, while the partition was much richer in the alpha 1-subunit than the alpha 2-subunit. Peptide mapping after limited proteolysis of tubulin subunits from the central pair, the wall of outer doublet microtubules except the partition, and the partition, indicated that alpha-subunits and beta-subunits from the former two sources were quite similar to each other, but distinguishable from the alpha-subunit (alpha 1') and beta-subunit of the partition. On the basis of these results, it can be said that at least three subspecies of alpha-subunit and two subspecies of beta-subunit exist in 'stable microtubules' found in the sperm flagellum. Under the electron microscope, the partition fraction contained ribbons consisting of two and three protofilaments. These results suggest that each of the two adjacent protofilaments constituting the partition is a thread of alpha 1' = beta dimers. In the partition, there were also five constitutive polypeptides in addition to tubulin.