The antimicrobial agent linezolid is approved for the treatment of severe infections caused by, e.g., methicillin-resistant Staphylococcus strains. In order to evaluate the penetration of linezolid into the interstitial space fluid (ISF) of subcutaneous adipose tissue and skeletal muscle of the target population, a microdialysis study was performed with 12 patients with sepsis or septic shock after multiple intravenous infusions. Unbound linezolid concentrations were determined for plasma and microdialysates by use of a validated high-performance liquid chromatography method. Individual compartmental pharmacokinetic (PK) analysis was performed using WinNonlin. In vivo microdialysis was found to be feasible for the determination of unbound linezolid concentrations at steady state in the ISF of critically ill patients. On average, linezolid showed good distribution into ISF but with high interindividual variability. A two-compartment model was fitted to unbound concentrations in plasma with a geometric mean distribution volume of 62.9 liters and a mean clearance of 9.18 liters/h at steady state. However, disposition characteristics changed intraindividually within the time course. In addition, an integrated model for simultaneous prediction of concentrations in all matrices was developed and revealed similar results. Based on the model-predicted unbound concentrations in ISF, a scheme of more-frequent daily dosing of linezolid for some critically ill patients might be taken into consideration to avoid subinhibitory unbound concentrations in the infected tissue. The developed integrated model will be a valuable basis for further PK data analysis to explore refined dosing guidelines that achieve effective antimicrobial therapy in all patients by use of the population PK approach.