Microtubule-associated-protein-dependent assembly of tubulin with GDP in the exchangeable site (tubulin-GDP) can occur with minimal free Mg2+ (< 3 microM). This reaction is totally inhibited by EDTA and by GTP concentrations over 2 mM and stimulated by MgCl2. Quantitative aspects of this stimulation are affected by both the Mg2+ and GTP concentrations but no relationship exists between reaction rates and relative amounts of different magnesium and GTP species. GTP binding to tubulin-GDP, while maximally stimulated 2-3-fold by exogenous MgCl2, was inhibited less than 50% by EDTA, and the amount of GTP bound increased as its concentration rose to levels that inhibited polymerization. Studies on the binding of Mg2+ to tubulin-GDP in the presence and absence of GTP showed that the increase in the amount of tubulin-associated Mg2+ was substoichiometric to the amount of GTP bound (maximum stoichiometry of additional Mg2+ to GTP bound, 0.7). Upon polymerization the increased Mg2+ content of tubulin was reduced, indicating its loss during GTP hydrolysis. Mg2+ thus plays a critical role in assembly distinct from its enhancement of GTP binding to the exchangeable site. If magnesium is present in trace amounts, this role must either be catalytic during polymerization or limited to nucleation.