To investigate safety and efficacy and identify the lowest effective dose of a new transdermal estradiol (E2) gel for relief of menopausal symptoms in a population of postmenopausal women. This study was a randomized, double-blind, placebo-controlled, multicenter, parallel-group study. Postmenopausal women with at least 60 hot flushes per week applied 0.87 g/d (n=136), 1.7 g/d (n=142), or 2.6 g/d (n=69) E2 gel or placebo gel (n=137) topically for 12 weeks. The changes from baseline in hot flush frequency and severity at 4 and 12 weeks and changes from baseline in vaginal atrophy symptoms at 12 weeks were examined. With increasing E2 doses, mean trough serum E2 increased from 17 to 29 pg/mL. By weeks 3-5, E2 gel reduced moderate-to-severe hot flush rate by at least seven hot flushes per day (P<.001) and reduced the severity score (P<.01). The numbers needed to treat for benefit for an 80% and 100% decrease in hot flush number were 3.2 and 6.3 for the 0.87-g/d group and 1.3 and 2.3 for the 2.6-g/d group. At week 12, vaginal pH was more acidic and vaginal maturation index more mature compared with placebo (P<.001). The lowest dose improved most bothersome vulvovaginal atrophy symptoms (P<.05). Estradiol gel was well tolerated at the site of application and produced no unexpected adverse effects. The 0.87 g/d dose produced fewest adverse events. The 0.87 g/d dose of this new transdermal E2 gel, which delivers an estimated 0.0125 mg E2 daily, delivered the lowest effective dose for treatment of vasomotor symptoms and vulvovaginal atrophy in a population of postmenopausal women. ClinicalTrials.gov, www.clinicaltrials.gov, NCT00391417. I.