The goal of this study was to determine the effect of acute transdermal 17-oestradiol (E-2) on the adipogenic potential of subcutaneous adipose-derived stem cells (ASC) in post-menopausal women. Post-menopausal women (n = 11; mean age 57 +/- 4.5 years) were treated for 2 weeks, in a randomized, cross-over design, with transdermal E-2 (0.15 mg) or placebo patches. Biopsies of abdominal (AB) and femoral (FEM) subcutaneous adipose tissue (SAT) were obtained after each treatment and mature adipocytes were analysed for cell size and ASC for their capacity for proliferation (growth rate), differentiation (triglyceride accumulation) and susceptibility to tumour necrosis factor alpha-induced apoptosis. Gene expression of oestrogen receptors and (ESR1 and ESR2), perilipin 1 and hormone-sensitive lipase (HSL), was also assessed. In FEM SAT, but not AB SAT, 2 weeks of E-2 significantly (P = 0.03) increased ASC differentiation and whole SAT HSL mRNA expression (P = 0.03) compared to placebo. These changes were not associated with mRNA expression of oestrogen receptors and , but HSL expression was significantly increased in FEM SAT with transdermal E-2 treatment. Adipose-derived stem cells proliferation and apoptosis did not change in either SAT depot after E-2 compared with placebo. Short-term E-2 appeared to increase the adipogenic potential of FEM, but not AB, SAT in post-menopausal women with possible implications for metabolic disease. Future studies are needed to determine longer term impact of E-2 on regional SAT accumulation in the context of positive energy imbalance.