In this study, paramagnetic centers over the cytosine were formed by photolysis then these centers were investigated using EPR method. EPR signals were not recorded from non-irradiated the cytosine, but irradiated polycrystalline exhibited complex EPR spectra. For obtaining of cytosine polycrystalline, novel crystallization method was performed on powder cytosine. Effective crystallization conditions were achieved by adjustment of the concentration of the metal ions, chemical solutions, NaCl, KCl, glacial asetic acid, nitric oxide, percloric acid, glutamic acid, and pH of buffer. Cytosine (C4H5N2O) polycrystalline obtained were irradiated with 60Co - rays at room temperature for 24 and 72 h. At the sample irradiated for 72 h, the paramagnetic centers were determined between 120 and 450 K by X-band EPR spectrometer. The spectra were found to be dependent slightly on temperature. Two cation radicals were determined in the structure and these were called Radicals I and II. The g and hyperfine constants were found to be aH2a = 61 G, aN2 = 9.39 G, a N1 = 7.15 G, and g1=2.0026 for the Radical I; a H3 = 10.57 G, aH1 = 3 G, aN3 = 6.72 G, a N1 = 5.36 G for, and g2=2.0034 the Radical II. © 2010 John Wiley & Sons, Ltd.