Based on the discovery that the estrogen synthase aromatase (CYP19A1) is abundantly expressed in high- grade gliomas, the aromatase inhibitor, letrozole is being investigated in pre-clinical models as a novel agent against this malignancy. Here, we investigated the systemic and brain pharmacokinetics of letrozole following single and steady state dosing in both male and female Sprague-Dawley rats. Furthermore, we employed physiologically-based pharmacokinetic (PBPK) modeling to gain quantitative insights into the blood-brain barrier penetration of this drug. Letrozole (4 mg/kg) was administered intraperitoneally daily for 5 days (for males) and 11 days (for females) and intracerebral microdialysis was performed for brain extracellular fluid (ECF) collection simultaneously with venous blood sampling. Drug levels were measured using HPLC and non-compartmental analysis was conducted employing WinNonlin®. Simcyp animal simulator was used for conducting bottom-up PBPK approach incorporating the specified multi-compartment brain model. Overall, marked gender-specific differences in the systemic and brain pharmacokinetics of letrozole were observed. Letrozole clearance was much slower in female rats resulting in markedly higher plasma and brain drug concentrations. At steady state, the plasma AUC 0-24 was 103.0 and 24.8 [mu]g*h/ml and brain ECF AUC 0-12 was 24.0 and 4.8 [mu]g*h/ml in female and male rats, respectively. The PBPK model simulated brain concentration profiles were in close agreement with the observed profiles. While gender-specific differences in letrozole PK are not observed in the clinical setting, these findings will guide the dose optimization during pre-clinical investigations of this compound. The PBPK model will serve as an important clinical translational tool.