Background: More than 60% of postmenopausal breast cancer (BC) patients have symptoms of dyspareunia and vaginal dryness that often result from endocrine therapy, and are challenging to treat. Local vaginal estrogens are proven safe and effective for VVA treatment, but a major concern of BC patients is the potential risk of systemic absorption and adverse breast effects; and it is contraindicated by the FDA to be used in this population. TX-004HR, an investigational, vaginal, softgel capsule of soluble 17β-estradiol (E2), is being developed to treat menopausal VVA. Serum E2 levels following TX-004HR treatment, which significantly reduced moderate-to-severe dyspareunia and vaginal dryness, were determined and compared with that of the normal postmenopausal range. Methods: A 12-wk, randomized, placebo-controlled, phase 3, safety/efficacy study (REJOICE) was conducted in menopausal women with VVA. TX-004HR (4 or 10 µg) was administered daily for 14 d, then twice weekly for 10 wks. Serum E2 levels using validated GC/MS/MS and pharmacokinetics (PK) were determined in 17-19 subjects/group on days 1 and 14 of daily dosing and day 84 of twice-weekly maintenance dosing. Results: The day 1, 0-h, mean±SD, serum E2 level was 4.05±2.69 pg/mL (95^th percentile 8.49 pg/mL). The mean 24-h average levels on day 1 for the placebo, 4 µg, and 10 µg groups were 4.86±3.22, 3.92±1.46, and 5.76±3.13 pg/mL, respectively. Day 14 mean serum levels were lower than those on day 1; 4.34±2.77, 3.63±1.78, and 4.59±2.27 pg/mL, respectively. No accumulation of E2 was observed on day 14. On day 84 (maintenance phase), serum E2 levels were 4.36, 4.25, and 4.79 pg/mL, respectively. Primary efficacy and safety endpoints had been met for both doses. Changes in E2 levels from baseline to day 14 and day 84 were similar. Conclusions: TX-004HR (4 or 10 µg) improved VVA symptomatology, while maintaining serum E2 levels within the normal postmenopausal range. Although not yet studied in BC patients, further study in this population, especially in those taking aromatase inhibitors, is warranted. Clinical trial information: NCT02253173