The cyclohexane and water permeability properties as well as the structure of the polyurethane-based pervaporation membranes were investigated. The polyurethanes (PU) were synthesised from poly(oxytetramethylene) diols (PTMO) of various molar masses, 2,4-tolylene diisocyanate (2,4-TD1), and different chain extenders used in various molar ratios. The microstructure of the synthesised PU was investigated by means of the density measurements, DSC method, and ESR technique with a spin probe TEMPO incorporated into PU via diffusion. The results show that only the ESR method was sensitive to all structural modifications applied enabling the correlations between the PU molecular structure and the mobility of the spin probe to be established. The diffusion coefficients (D) of cyclohexane and water were calculated based on the pervaporation results and the sorption data, and were correlated with the rotation correlation time (tau) of the spin probe as a measure of the microstructure of the permeable domains. The exponential and linear empirical equations were obtained for the PU/cyclohexane and PU/water systems, respectively, as a result of the fitting procedure applied. These correlations demonstrate that a spin probe method might be useful to characterise free volume, especially in case of complex membrane materials, and to predict the flux of permeant through the pervaporation membranes. (C) 2004 Elsevier Ltd. All rights reserved.