Hop-derived food supplements and beers contain the prenylflavonoids xanthohumol (X), isoxanthohumol (IX) and the very potent phyto-oestrogen (plant-derived oestrogen mimic) 8-prenylnaringenin (8-PN). The weakly oestrogenic IX can be bioactivated via O-demethylation to 8-PN. Since IX usually predominates over 8-PN, human subjects may be exposed to increased doses of 8-PN. A dietary intervention trial with fifty healthy post-menopausal Caucasian women was undertaken. After a 4 d washout period, participants delivered faeces, blank urine and breath samples. Next, they started a 5 d treatment with hop-based supplements that were administered three times per d and on the last day, a 24 h urine sample was collected. A semi-quantitative FFQ was used to estimate fat, fibre, alcohol, caffeine and theobromine intakes. The recoveries of IX, 8-PN and X in the urine were low and considerable inter-individual variations were observed. A five-fold increase in the dosage of IX without change in 8-PN concentration resulted in a significant lower IX recovery and a higher 8-PN recovery. Classification of the subjects into poor (60%), moderate (25%) and strong (15%) 8-PN producers based on either urinary excretion or microbial bioactivation capacity gave comparable results. Recent antibiotic therapy seemed to affect the 8-PN production negatively. A positive trend between methane excretion and 8-PN production was observed. Strong 8-PN producers consumed less alcohol and had a higher theobromine intake. From this study we conclude that in vivo O-demethylation of IX increases the oestrogenic potency of hop-derived products.