Single crystals of ergosterol were investigated by Electron Paramagnetic Resonance (EPR) technique, with γ irradiation of the crystals at different orientations in the magnetic field between temperatures of 120 and 380 K, and the spectra were found to be slightly dependent on temperature. Because of the importance of ergosterol it is important to determine the irradiation effects on this molecule. Taking into consideration the chemical structure and the experimental spectra of the irradiated single crystal ergosterol, we found that two paramagnetic species which were labeled as radical A, CH 2αH β, and radical B, CH αH βH γH σ, were produced in the host crystal. The EPR parameters; spectroscopic splitting factor, g, and hyperfine coupling constant, a, were determined for each radical. © 2011 Elsevier B.V. All rights reserved.