Microdialysis in freely moving rats coupled to high-performance liquid chromatography (HPLC) was used to measure the free concentration of acetaminophen (APAP) in blood and cerebrospinal fluid (CSF) after an intravenous bolus dose (25 mg/kg). In vitro calibration of two commercially available probe types was performed in 0.9% NaCl solution and blood. The influence of these media on recovery was tested by retrodialysis. This technique was also used for in vivo calibration and to monitor the dynamics of the performance of implanted probes. The results were compared with data obtained from conventional sampling techniques of direct withdrawal of blood and CSF, and also with the results obtained by correcting dialysate concentrations using in vitro recovery values. The data demonstrate that whole blood lowers recovery not only by reducing the free concentration of drug, but also by directly influencing dialysis efficiency (mean reduction of recovery: 50.1%). By contrast, low transport capacities of CSF surrounding the implanted probe lead to suboptimal conditions and, therefore, to a reduction of in vivo recovery (mean reduction of recovery: 65.5%). After correction of recovery values using in vivo retrodialysis prior to dosing the animal, we obtained similar data as compared to conventional sampling techniques. These results demonstrate that microdialysis may provide a minimally invasive method to monitor the free concentrations of drugs, such as acetaminophen, in different compartments, and allow a multitude of pharmacokinetic data to be obtained from freely moving animals.