The effect of synaptic junction (SJ) on microtubule assembly was examined. After preincubation with ATP at 37 degrees C, rat SJ decreased the initial velocity and the extent of the porcine brain microtubule assembly (initiated by the addition of GTP) in a Ca2+/calmodulin (CaM)-dependent manner. The degree of the inhibition reached 35% of the control assembly (0-min preincubation) after 20-min preincubation with ATP. There was no inhibition either with heat-treated SJ, at 0 degree C, or in the presence of EGTA or W-7 (CaM antagonist). The inhibition was due neither to protease(s) nor CaM contaminating the preparations. Free Ca2+ concentration level required for the inhibition of microtubule assembly was 10(-6)M. Phosphorylation of microtubule proteins was inhibited by SJ in a Ca2+/CaM-dependent manner, and the inhibition occurred in a physiological increase range of intracellular Ca2+ concentration (10(-6)M). The heat-treated SJ caused no inhibition. The result suggested that the microtubule assembly in the postsynaptic region was regulated by a Ca2+/CaM-dependent protein kinase associated with SJ; i.e., major postsynaptic density protein.