Calcium ions can inhibit the in vitro assembly of microtubules and, therefore, may play a role in the regulation of microtubule formation in vivo. In order to test the validity of this hypothesis; the interaction between calcium and pruified brain microtubular protein has been investigated by standard binding assays. We have detected and characterized two classes of binding sites for calcium on tubulin, the major component of cytoplasmic microtubules. There is a single high-affinity site per tubulin molecule, characterized by a dissociation constant of 3.2 X 10(-6) M. That site is inhibited by magnesium (k1 = 5 X 10(-5) M) and potassium chloride. There are approximately 16 low-affinity sites which have a dissociation constant of 2.8 X 10(-4) M, and which are also inhibited by potassium chloride. Binding at the low-affinity sites is slightly enhanced by low magnesium concentrations. Both classes of sites are distinguishable from the colchicine binding site, and are apparently also distinct from the vinblastine and guanine nucleotide sites. The characteristics of the calcium binding activity of tubulin are similar to those found for the calcium-binding proteins of sarcoplasmic reticulum. The results are consistent with a physiological role for calcium in the regulation of microtubule assembly.