Oxazolidinones are a novel class of antibacterials with excellent activity against resistant Gram-positive bacteria including strains causing multidrug-resistant tuberculosis (TB). Despite their excellent efficacy, optimal dosing strategies to limit their toxicities are still under development. Here, we developed a novel synthetic strategy for fluorine-18-radiolabeled oxazolidinones. As proof-of-concept, we performed whole-body 18F-linezolid positron emission tomography (PET) in a mouse model of pulmonary TB for noninvasive in situ measurements of time-activity curves in multiple compartments with subsequent confirmation by ex vivo tissue gamma counting. After intravenous injection, 18F-linezolid rapidly distributed to all organs with excellent penetration into Mycobacterium tuberculosis-infected lungs. Drug biodistribution studies with PET can provide unbiased, in situ drug measurements, which could boost efforts to optimize antibiotic dosing strategies.