Using the techniques of FTIR-microscopy and cryomicrotomy, the diffusion behavior of a model solute, 4-cyanophenol, through PHEMA hydrogel membranes was observed. In a side-by-side membrane permeation study, the experiment was stopped before or when the steady state was achieved. The membrane was removed from the experimental device, freeze-dried, and sectioned using a cryomicrotome. The solute concentration profile across the membrane could be determined by an FTIR-microscope. Based on Fick's law and the solution-diffusion model, the diffusion coefficient of the solute in a membrane permeation study can be determined from the pseudo-steady state permeation rate, time-lag method, and the concentration profiles within the membrane in the transient and pseudo-steady states. The results showed that the diffusion coefficients determined by different methods were not the same and distributed from 8.9 x 10(-9) to 1.7 x 10(-7) cm(2)/s. The variation in diffusion coefficients was most likely due to the strong interaction between 4-cyanophenol and PHEMA. Some portion of absorbed 4-cyanophenol was immobilized on the polymer chains or trapped by the networks so that it was not able to diffuse. As a result, the diffusion coefficients obtained from different methods may represent different physical meanings and need a careful justification. (c) 2005 Elsevier B.V. All rights reserved.