Salicylic acid (SA) can inhibit the facilitated transport of inorganic sulfate in the kidney, placenta, and erythrocytes. One mechanism of this inhibition could involve the interaction of SA with membranes, resulting in altered function of transporter protein(s) due to changes in membrane fluidity. Such membrane effects could result in altered membrane transport and consequently in changes in the pharmacokinetics and the therapeutic activity of both xenobiotics and endogenous substrates. We investigated the effect of SA on the fluidity of brush border membrane (BBM) and basolateral membrane (BLM) isolated from rat kidney and also on the physical properties (such as phase transition temperature and fluidity) of model membranes by fluorescence polarization and differential scanning calorimetry (DSC) techniques. SA decreased the lipid order parameter (S) of BBM and BLM membranes in a concentration-dependent manner, indicating that the addition of SA makes the membrane more fluid. The fluidizing effect of SA was more pronounced than that of benzyl alcohol. Studies were carried out with protein-free model membranes composed of dipalmitoylphosphatidylcholine (DPPC) to investigate the effects of SA on the bilayer membrane lipids. SA decreased the fluorescence polarization of DPH (1,6-diphenyl 1,3,5-hexatriene) incorporated in DPPC vesicles. DSC studies demonstrated that SA broadened the phase transition temperature of DPPC vesicles and suggested that SA is located in the C1-C8 region of the acyl chain. In protein-free model membranes, SA exerted fluidizing effects through its incorporation into the cooperative hydrophobic region of the bilayer. The perturbation of membrane physical properties induced by SA and its hydrophobic localization in the membrane bilayer may be important in the SA-induced alteration of sulfate membrane transport.