Free drug assay in physiological fluids is getting more and more attention nowadays. The principal reason is the fact that the unbound drug form is responsible for the therapeutic or toxic effects of its application. Unbound drug concentration significantly depends on the extent of its binding by plasma. This article describes the influence of different factors on unbound propofol concentration. These factors are presence of infusion fluids in blood, type of anticoagulant, sample storage time and plasma freezing. The following conclusions result from the experiments carried out: 1. The lowest free drug fractions are observed in samples containing carbohydrate infusion fluids. The free drug percentage is virtually independent of its total concentration in the range of clinically relevant concentrations. 2. There is no evident anticoagulant influence (heparin, citrate, EDTA and oxalate) on free propofol level in plasma samples. 3. Longer storage of plasma at 4 degrees C causes a slight rise of free propofol concentration in heparinised plasma and no evident changes in plasma containing citrate. 4. Plasma freezing induces the increase of free drug concentration both for citrate and heparin. These findings are valuable both for clinicists and pharmacologists, and important for chemical analysts.