We have reported earlier the purification of tubulin from a plasmalemmal-microsomal fraction derived from rat hippocampus using an estradiol (E<sub>2</sub>) affinity column and the specific binding of tubulin to both E<sub>2</sub> and testosterone (T). To further investigate the effect of E<sub>2</sub> and T on the function of this protein, changes in microtubule polymerization as a result of exposure to the steroids were examined in this study, using both pure tubulin and rat hippocampal primary cell cultures. First, pure tubulin was incubated with or without steroids for 30 min on ice followed by polymerization at 37°C. The numbers of microtubules formed were counted from electron microscopic pictures. The results showed that at 30 min of polymerization, 10 n M, 30 n M and 30 µ M of E<sub>2</sub> inhibited microtubule assembly by –70%, –94%, and –92%, respectively (p < 0.01), while T at the same three concentrations stimulated it by +83%, +66%, and +121%, respectively (p < 0.05). The inhibitory effect of E<sub>2</sub> and the stimulatory effect of T were observed at 15, 30 and 60 min of the polymerization process. Next, primary cell cultures from 17-day rat fetus hippocampal tissues were treated with the steroids and polymerized microtubules (Triton X-100 resistant) were examined by immunocytochemistry. The results demonstrated that 60 min of E<sub>2</sub> treatment (10 n M) decreased the intensity of the immunolabeling of polymerized microtubules. The effect of T at n M concentration was not significant though it increased the immunolabeling at µ M concentration. Of great significance was a remarkable inhibition by T of the well-established depolymerization effect of colchicine in both the pure tubulin assay and the cell culture model, while E<sub>2</sub> was not effective. In an effort to pursue the possible mechanism(s) of the effect of E<sub>2</sub> and T on microtubule formation, we found that T only inhibited the microtubule depolymerization process without affecting the rate of polymerization. In contrast, E<sub>2</sub> modifies only the polymerization process without altering the depolymerization. Overall, these data indicate that E<sub>2</sub> and T may be considered as novel regulators of microtubule dynamics and thereby controlling cytoskeleton function in cells.