AbstractHop‐derived products may contain xanthohumol (XN), isoxanthohumol (IX), and the potent phytoestrogen 8‐prenylnaringenin (8‐PN). To evaluate the potential health effects of these prenylflavonoids on breast tissue, their concentration, nature of metabolites, and biodistribution were assessed and compared with 17β‐estradiol (E2) exposure. In this dietary intervention study, women were randomly allocated to hop (n=11; 2.04 mg XN, 1.20 mg IX, and 0.1 mg 8‐PNpersupplement) or control (n=10). After a run‐in of ≥4 days, three supplements were taken daily for 5 days preceding an aesthetic breast reduction. Blood and breast biopsies were analyzed using HPLC‐ESI‐MS/MS. Upon hop administration, XN and IX concentrations ranged between 0.72 and 17.65 nmol/L and 3.30 and 31.50 nmol/L, and between 0.26 and 5.14 pmol/g and 1.16 and 83.67 pmol/g in hydrolyzed serum and breast tissue, respectively. 8‐PN however, was only detected in samples of moderate and strong 8‐PN producers (0.43–7.06 nmol/L and 0.78–4.83 pmol/g). Phase I metabolism appeared to be minor (∼10%), whereas extensive glucuronidation was observed (> 90%). Total prenylflavonoids showed a breast adipose/glandular tissue distribution of 38/62 and their derived E2‐equivalents were negligible compared with E2in adipose (384.6±118.8 fmol/g,p=0.009) and glandular (241.6±93.1 fmol/g,p<0.001) tissue, respectively. Consequently, low doses of prenylflavonoids are unlikely to elicit estrogenic responses in breast tissue.