We wanted to investigate the effects of gamma radiation on DNA single-strand breaks (SSB) and glutathione (GSH) levels in mononuclear blood cells (MNC) of radiotherapy technicians. DNA SSB in MNC of radiotherapy technicians who use Co-60-gamma source in their works were detected by alkaline filter elution and compared to control subjects. In addition, GSH levels were measured using the enzymatic method in MNC. Blood samples were collected from radiotherapy technicians on Monday and Friday. DNA SSB levels were found to be significantly higher in smoking controls compared to non-smoking controls. Significant increases of 36% and 49% in DNA SSB were detected from Monday to Friday for non-smoking and smoking radiotherapy technicians, respectively. GSH levels were found to be decreased significantly from Monday to Friday. gamma-Radiation resulted in increased DNA SSB levels of MNC in radiotherapy technicians throughout the working week and these breaks have been observed to be repaired at the weekend. Smoking habit caused an additional increase in the SSBs observed in radiotherapy technicians.