Background And Objectives Because of extensive first‐pass metabolism, oral bioavailability of sildenafil reaches only 40%. Formation of the primary metabolite, N‐desmethylsildenafil, is mainly mediated by the cytochrome P450 enzyme CYP3A4. In this study we investigated the influence of grapefruit juice, containing inhibitors of intestinal CYP3A4, on the pharmacokinetics of sildenafil and N‐desmethylsildenafil. Methods In a randomized crossover study, 24 healthy white male volunteers received single 50‐mg doses of sildenafil. Two doses each of 250 ml grapefruit juice or water, respectively, were administered 1 hour before and together with the drug. Plasma concentrations of sildenafil and N‐desmethylsildenafil were determined up to 24 hours post dose by use of liquid chromatography–tandem mass spectrometry (limit of quantification, 1 ng/ml). Results Grapefruit juice changed the area under the sildenafil plasma concentration–time curve from time zero to infinity AUC(0‐∞) from 620 1.53 ng/ml • h to 761 1.58 ng/ml • h (geometric mean with geometric standard deviation), corresponding to a 23% increase (90% confidence interval, 13%‐33%). N‐Desmethylsildenafil AUC(0‐∞) increased by 24% (90% confidence interval, 17%‐32%). Maximum plasma concentrations (Cmax) of sildenafil and N‐desmethylsildenafil were essentially unchanged. There was a trend toward a prolonged time to reach Cmax during the grapefruit juice period (from a median of 0.75 hour to a median of 1.13 hours), corresponding to an increase by 0.25 hour (90% confidence interval, 0–0.63 hour). Interindividual variability was pronounced in both periods. Conclusions Grapefruit juice increases sildenafil bioavailability and tends to delay sildenafil absorption. Sildenafil pharmacokinetics may become less predictable with grapefruit juice. Although patients usually will not be endangered by concomitant use of grapefruit juice, it seems advisable to avoid this combination. Clinical Pharmacology & Therapeutics (2002) 71, 21–29; doi: 10.1067/mcp.2002.121236