The knowledge about the effect of estradiol on tendon connective tissue is limited. Therefore, we studied the influence of estradiol on tendon synthesis, structure and biomechanical properties in postmenopausal women. Non-users (Control, n=10) or habitual users of oral estradiol replacement therapy (ERT, n=10) were studied at rest and in response to one-legged resistance exercise. Synthesis of tendon collagen was determined by stable isotope incorporation (fractional synthesis rate (FSR)) and microdialysis technique (N-terminal propeptide of type I collagen synthesis, PINP)). Tendon area and fibril characteristics were determined by MRI and transmission electron microscopy, whereas tendon biomechanical properties were measured during isometric maximal voluntary contraction by ultrasound recording. Tendon FSR was markedly higher in ERT-users (P<0.001), whereas no group difference was seen in tendon PINP (P=0.32). In ERT-users positive correlations between s-estradiol and tendon synthesis were observed, whereas change in tendon synthesis from rest to exercise was negatively correlated to s-estradiol. Tendon area, fibril density, fibril volume fraction and fibril mean area did not differ between groups. However the percentage of medium size fibrils was higher in ERT-users (P<0.05), whereas the percentage of large fibrils tended to be greater in Control (P=0.10). A lower Youngs modulus (GPa/%) was found in ERT-users (P<0.05). In conclusion, estradiol administration was associated with higher tendon FSR and a higher relative number of smaller fibrils. Whereas this indicates stimulated collagen turnover in the resting state, collagen responses to exercise were negatively associated with s-estradiol. These results indicate a pivotal role for estradiol in maintaining homeostasis of female connective tissue.