Background & Objective: Estradiol and misoprostol have been used for the enhancement of Transformation Zone (TZ) visibility in vaginal colposcopy. However, no consensus has been reached on the superiority of one medication over the other. This study aimed to compare the efficacy of estradiol and misoprostol for the enhancement of TZ visibility in vaginal colposcopy of premenopausal and postmenopausal women. Materials & Methods: In this clinical trial, 78 patients with unsatisfactory colposcopy were randomly divided into three groups. Group 1 (n=25) received 25 µ, g of vaginal estradiol for 14 days prior to colposcopy. Group 2 (n=27) received 400 µ, g of misoprostol 12 h prior to colposcopy. Group 3 (n=26) served as the control group and did not receive any medication. Visibility of the TZ, age, body mass index (BMI), history of vaginal delivery, history of sexually transmitted diseases, history of human papillomavirus (HPV), the reason for colposcopy, and drug-related side effects were compared among the three groups and also between premenopausal and postmenopausal women. Data were analyzed using analysis of variance (ANOVA), Kruskal-Wallis, Chi-square, and Fisher’, s exact tests. Results: The percentage of TZ visibility was 72%, 55. 6%, and 26. 9% in the estradiol, misoprostol, and control groups, respectively (P=0. 005). These values were 70%, 33. 3%, and 0%, respectively, in postmenopausal women (P=0. 043) and 60%, 72. 7%, and 33. 3%, respectively, in premenopausal women (P=0. 152). Regarding drug-related side effects, there was no statistically significant difference between the three groups (P=0. 374). Conclusion: Estradiol was significantly superior to misoprostol for the enhancement of TZ visibility, particularly in postmenopausal women, with no difference regarding side effects.