Limited proteolysis of the tubulin dimer (β£β€) by subtilisin occurs more rapidly with β€ than with β£ tubulin. This leads to the formation of an intermediate hybrid dimer, β£β€ s , before both C termini are cleaved to form tubulin S(β£ s β€ s ). The three forms of tubulin usually coexist in subtilisin-treated preparations and such cross-contamination can be reliably detected only by running SDS-polyacrylamide gels well beyond expulsion of the dye front. Previously published preparations have not ruled out such contamination or have formed poorly reversible polymers. Because ion exchange separation incurred substantial protein losses, we have developed a new protocol for rapid preparation of tubulin S (β£ s β€ s , free of β£β€ or β£β€ s ) that is based on proteolysis at low ionic strength. This increases the relative rate of C terminal cleavage of β€ tubulin. The product forms sheets, bundles, or rings that are depolymerized by cold, salt, and podophyllotoxin, partially depolymerized by Ca 2Ο© , and has a decreased critical concentration for polymerization that can be further decreased by taxol. We have also found a method for forming nearly pure β£β€ s dimers by using methods that retard proteolysis of the C terminus of β£ tubulin.