We examined the effect of porcine brain Ca2+-dependent regulator (CDR) protein on microtubule (MT) assembly from microtubular proteins isolated from porcine brain by temperature-dependent cycles of assembly-disassembly. CDR exhibited a potent inhibitory effect on MT assembly in the presence of Ca2+, whereas it had little or no effect on the extent of MT assembly in the absence of Ca2+. The increase in KCl concentration greatly potentiated the Ca2+-dependent inhibitory effect of CDR. The effect of CDR was reversible in a Ca2+ concentration-dependent manner, and the extent of inhibition by CDR at a fixed concentration of free Ca2+ was roughly proportional to the concentration of CDR. Moreover, the Ca2+ concentration required for the half-maximal inhibition of MT assembly from a fixed concentration of purified microtubular proteins (PMP) decreased with increasing CDR concentration. On the basis of these results, together with data on the Ca2+-dependent association of CDR and tubulin (J. Biochem., accompanying paper), we propose the following model; Ca2+ + CDR in equilibrium Ca2+-CDR Ca2+-CDR + tubulin in equilibrium Ca2+-CDR-tubulin (nonpolymerizable).