Abstract Recent observations indicate that pharmacokinetics of β-lactam antibiotics in the lung can be predicted by the use of concentration versus time profiles in peripheral soft tissues. If this observation is transferred to other classes of antimicrobials, measurement of antimicrobial concentrations in peripheral tissues would enable prediction of the pharmacokinetics of antimicrobials at the site of the respiratory tract infection. We set out to test the hypothesis that concentrations of the fluoroquinolone levofloxacin in the respiratory tract can be predicted on the basis of knowledge of its pharmacokinetics in peripheral soft tissues. After administration of a single intravenous dose of 500 mg of levofloxacin, microdialysis was used to describe the concentration versus time profiles of levofloxacin in the interstitial space fluid of lung tissue of patients ( n = 5) undergoing elective lung surgery. These data were compared with the concentration versus time courses in the interstitial space fluid of skeletal muscle and subcutaneous adipose tissue of healthy volunteers ( n = 7). The median AUC 0–∞ of free levofloxacin in lung (2267 mg × min/L, 1980–2355) was about 2-fold and 1.5-fold lower compared with skeletal muscle (4381 mg × min/L, range 1720–8195) and adipose tissue (3492 mg × min/L, range 1323–6420) of healthy controls, respectively. Concentrations in the interstitial space fluid of the lung were descriptively lower compared with corresponding concentrations in peripheral soft tissues. This is in contrast to previous observations made for the class of β-lactam antibiotics, and indicates that pharmacokinetics of levofloxacin derived from soft tissues may not be used uncritically for prediction of levofloxacin concentrations in the interstitium of the lung.