We determined lung bioavailability of a fluticasone propionate (FP) pressurised metred-dose inhaler (Flovent HFA; GlaxoSmithKline, Research Triangle Park, NC, USA) administered via AeroChamber Plus (Monaghan Medical, Plattsburgh, NY, USA) with Facemask and Babyhaler (GlaxoSmithKline) valved holding chambers (VHCs) using a population pharmacokinetic approach. Children from 1 to <4 yrs of age with stable asthma but a clinical need for inhaled corticosteroid therapy were administered 88 μg FP hydrofluoroalkane (2 × 44 μg) twice daily delivered through the two devices in an open-label, randomised crossover manner for 8 days each. Patients were randomised to one of three sparse sampling schedules for blood collection throughout the 12-h dosing interval on the 8th day of each treatment for pharmacokinetic analysis. The area under the FP plasma concentration-time curve (AUC) was determined for each regimen. 17 children completed the study. The population mean AUC following FP with AeroChamber Plus with Facemask was 97.45 pg·h·mL(-1) (95% CI 85.49-113.32 pg·h·mL(-1)) and with Babyhaler was 51.55 pg·h·mL(-1) (95% CI 34.45-64.46 pg·h·mL(-1)). The relative bioavailability (Babyhaler/AeroChamber Plus) was 0.53 (95% CI 0.30-0.75). Clinically significant differences in lung bioavailability were observed between the devices. VHCs are not interchangeable, as differences in drug delivery to the lung may occur. A population pharmacokinetic approach can be used to determine lung bioavailability of FP.