Background: To investigate the correlation between CYP3A4/5 activity and clarithromycin metabolism, and between CYP3A activity and CYP3A genotype. Methods: This is an open-label, prospective pharmacokinetic study evaluating CYP3A activity using The Erythromycin Breath Test. Eight blood samples were collected within 12. h after clarithromycin 500. mg was administered orally. The clarithromycin concentrations were measured by liquid chromatography-tandem mass spectrometry. AUC, Tmax and Cmax were calculated. Selected Single Nucleotide polymorphisms in CYP3A4/5 genes were assessed by PCR and single base extension. Results: Twenty-one chronically infected patients were included. An 8-fold variation in the CYP3A4 activity, 10-fold variation in AUC for clarithromycin (median 881 μg/mL × min), and a 16-fold variation in Cmax for clarithromycin (median 3.4. μg/mL) were found. A linear correlation between the CYP3A4-activity and clarithromycin metabolism was demonstrated (P < 0.05). Conclusion: The large variation in the clarithromycin pharmacokinetics in cystic fibrosis patients may cause treatment failure. The Erythromycin Breath Test could be valuable in identifying cystic fibrosis patients in risk of treatment failure/drug toxicity.