Objective ur objective was to assess the contribution of 6′,7′‐dihydroxybergamottin (DHB) to the inhibitory effect of grapefruit juice toward intestinal cytochrome P450 (CYP) 3A4. Methods An aqueous extract was prepared from grapefruit juice by centrifugation, filtration, and repeated washing of the particulate with water. The concentrations of various furanocoumarins in this grapefruit juice “serum” and in whole grapefruit juice were measured by HPLC and their identities confirmed by liquid chromatography–tandem mass spectrometry. Five healthy volunteers were given a single tablet of felodipine (10 mg) with whole grapefruit juice, orange juice–containing serum, or plain orange juice (control). The pharmacokinetic outcomes of felodipine were evaluated by noncompartmental methods. The effects of serum and purified DHB (at the same concentrations as those measured in the orange juice–containing serum used in the clinical study) were compared, in vitro, with regard to (1) the reversible and mechanism‐based inhibition of the catalytic activity of complementary deoxyribonucleic acid–expressed CYP3A4 and (2) the time‐dependent loss of immunoreactive CYP3A4 protein in modified Caco‐2 cells. Results The concentration of DHB in serum was comparable to that measured in whole grapefruit juice (38 μmol/L versus 43 μmol/L), and the concentrations of other known furanocoumarins were well below the lowest published concentration required to inhibit catalytic activity by 50%. Relative to plain orange juice, orange juice–containing serum significantly increased the median felodipine area under the plasma concentration–time curve by 1.9‐fold (P = .04) and increased the maximum concentration by 1.7‐fold (P = .01). In vitro, serum and purified DHB had similar inhibitory effects toward CYP3A4 activity with respect to both reversible inhibition (95% confidence interval, 85% ± 5.7% and 75% ± 4.5%, respectively) and mechanism‐based inhibition after a 15‐minute preincubation (95% confidence interval, 79% ± 6.8% and 78% ± 5.7%, respectively). In Caco‐2 cells the time‐averaged extents of CYP3A4 protein loss caused by serum and purified DHB were identical (43%). Conclusion The interaction between grapefruit juice serum and felodipine can be attributed largely to DHB. This establishes DHB as an important contributor to the grapefruit juice effect. Clinical Pharmacology & Therapeutics (2004) 75, 569–579; doi: 10.1016/j.clpt.2004.02.007