Abstract Context High mammographic density in postmenopausal women is an independent risk factor for breast cancer by undetermined mechanisms. No preventive therapy for this risk group is available. Activated platelets release growth factors that modulate the microenvironment into a protumorigenic state. Estrogens may affect the risk of breast cancer and platelet function. Whether platelets are activated in situ in breast cancer or in normal breast tissue at high risk of breast cancer and the association to estradiol remains elusive. Objective To investigate whether platelets are activated in situ in breast cancers and in dense breast tissue of postmenopausal women and explore correlations between estradiol, released platelet factors, and inflammatory proteins. Setting and design Sampling of in vivo proteins was performed using microdialysis in a total of 71 women: 10 with breast cancer, 42 healthy postmenopausal women with different breast densities, and 19 premenopausal women. Results Our data demonstrate increased levels of coagulation factors in dense breast tissue similar to that found in breast cancers, indicating excessive platelet activation. Premenopausal breasts exhibited similar levels of coagulation factors as postmenopausal dense breasts. Out of 13 coagulations factors that were upregulated in dense breasts, 5 exhibited significant correlations with estradiol, both locally in the breast and systemically. In breast tissue, positive correlations between coagulation factors and key inflammatory proteins and matrix metalloproteinases were detected. Conclusions Breast density, not estradiol, is the major determinant of local platelet activation. Inactivation of platelets may be a therapeutic strategy for cancer prevention in postmenopausal women with dense breasts.