Since its extensive development in the early 1980s, SFE has attracted considerable attention as a sample-preparation procedure. However, other different sample preparation procedures, including precipitation, liquid- and/or solid-phase extraction in biological fluids, also remain in use. In this investigation, SFE was introduced to isolate and identify orbifloxacin from plasma and milk. Four parameters, including the temperature and the pressure of supercritical fluid, modifier ratios, and dynamic extraction time, were evaluated and optimized to obtain the best yield of the analyte from the biological fluids. Determinations of the orbifloxacin (OBFX) in the extracts were carried out using HPLC-FLD. The optimum conditions of the extraction process that yielded the maximum analyte extraction efficiencies were 150 degrees C vs. 60 degrees C, 250 kg cm(-2), 30% vs. 35% methanol, and 40 min vs. 20 min, for plasma and milk, respectively. The linearity of the calibration curves as well as the instrument LODs/LOQs were evaluated. Good linearity (at least r(2) > or = 0.999) of the calibration curves was obtained over the range from 0.2 to 0.01 microg mL(-1). The method showed a good recovery rate (74.2-127.73%) and precision (RSDs: 1.64-20%). The instrumental LOD and LOQ values were 0.004 microg mL(-1) vs. 0.01 microg mL(-1) or 0.006 microg mL(-1) vs. 0.02 microg mL(-1), for plasma and milk, respectively. The method was successfully applied to estimate the pharmacokinetic variables of orbifloxacin in lactating does. To the best of our knowledge, this is the first time that SFE has been applied to isolate an antimicrobial agent from biological fluids. This method is promising for clinical applications and for pharmacokinetic studies of various pharmaceuticals in biological fluids.