BACKGROUND Levonorgestrel a synthetic progestagen used for endometriosis, dysmenorrhea and emergency contraception, is quickly and completely absorbed in the digestive tract. levonorgestrel is predominantly metabolised through hepatic routes that utilise the CYP3A system (CYP3A4 and CYP3A5). OBJECTIVE This study aimed to evaluate the association between variant alleles of CYP3A4*1B and CYP3A5*3 polymorphisms and the pharmacokinetics of levonorgestrel. METHODS A group of 17 adult female healthy volunteers who signed an informed consent were genotyped for CYP3A4 and CYP3A5 through PCR-RFLP. Volunteers were submitted to pharmacokinetic analysis where, after a 12-hour overnight fast, they received a single oral dose of 0.75 mg of levonorgestrel. Serial blood samples were obtained (0 to 24 hours), and levonorgestrel concentrations were determined by UPLC-MS/MS to determine pharmacokinetic parameters. The procedures employed herein were performed according to the Declaration of Helsinki and Good Clinical Practices standards. RESULTS Observed genotype frequencies in the studied group for CYP3A4*1B were 11.8% for *1B/*1B, 5.8% for *1/*1B and 82.4% for *1/*1. CYP3A5*3 frequencies were 70.5% for *3/*3, 23.5% for *1/*3 and 6.5% for *1/*1. A high pharmacokinetic variability between volunteers was observed, but no statistical association of pharmacokinetic parameters was found within the studied CYP3A4/5 polymorphisms. CONCLUSIONS Genetic polymorphisms could be important factors in determining inter-patient variability in plasma levonorgestrel concentrations, which in this study were not significantly associated with the presence of CYP3A4*1B and CYP3A5*3 polymorphisms. Therefore, due to the significant inter-patient variability that we observed during the course of this study, it is necessary to carry out studies with larger number of volunteers.