Estrogen receptor (ER) testing of breast cancer imperfectly predicts response to endocrine therapy (ET). We hypothesize that a brief estradiol challenge will increase tumor progesterone receptor (PgR) levels only in tumors with functional ER. In this prospective, phase 2, single-center, single-arm trial (NCT02455453), we report the association of response to ET with change in tumor uptake of the progestin analog, 21-[18F]fluorofuranylnorprogesterone (FFNP), before and after a one-day estradiol challenge. In 43 postmenopausal women with advanced ER+ breast cancer, we show a post-challenge increase in tumor FFNP uptake only in 28 subjects with clinical benefit from ET (responders), but not in 15 without clinical benefit (nonresponders) (p < 0.0001), indicating 100% sensitivity and specificity. We further show significantly longer survival (p < 0.0001) in the responding subjects. Our results demonstrate that change in tumor FFNP uptake after estradiol challenge is highly predictive of response to ET in women with ER+ breast cancer.