Electron paramagnetic resonance (EPR) spectroscopy was applied to the study of fluidity in thylakoids of durum wheat (Triticum durum Desf. cv. Creso) treated with oxygen free radicals. The radicals were generated by using Fe2+-EDTA and ascorbic acid and let to act either on thylakoid membranes isolated from leaves or on leaves themselves, from which thylakoids were subsequently extracted. The oxidative treatment was prolonged for periods ranging from 15 min to 3 h. Line shape analysis of EPR spectra recorded as a function of temperature on concentrated suspensions of thylakoids labeled with 5-doxylstearic acid (5-DSA) allowed to get information about the fluidity of differently treated membranes. An axially symmetric rotational diffusion model, defined by the components of the rotational tenser D-par and D-perp and by the diffusion tilt angle theta, was assumed for the dynamics of the spin probes. Good agreement with experimental spectra was obtained with a diffusion tilt angle continuously increasing with temperature and a high anisotropy ratio N = D-par/D-perp of the spin probe rotational diffusion. High N values have been considered as an indication for the immobilization of the spin probes in the hydrophobic part of the membranes. The changes of theta with temperature have been interpreted in terms of chain isomerization within the probes. The differences found between the probe dynamics in the differently treated thylakoids have been discussed on the basis of the time and type (on the leaves or on the membranes) of oxidative treatment.