The overexpression of eNOS cell line was constructed to investigate the role of eNOS in the induction of radiation induced bystander effects without pharmacologic intervention. By using of the fluorescent probe DAF-FM and Griess reagent system, the formation of the extracellular and intracellular NO in irradiated donor cell (HEK-Vector and HEK-eNOS) was detected indirectly. DNA double strand breaks and micronucleus were measured in the recipient MRC5 cells after the MRC5 was co-cultured with the condition medium from the irradiated donor cells and the irradiated cells themselves, respectively. The result showed that the levels of both extracellular and intracellular NO elevated at peak at 15 min after exposure to X-rays. The medium of the irradiated donor cells at 15 min after exposure to 5 Gy X-rays was transferred to recipient cells and incubated for 30 min. More bystander γ H2AX foci, a biomarker of DSB, were observed at 15 min in the bystander cells co-cultured with the condition medium from the irradiated eNOS overexpression cells. Significant increment of MN was found in the bystander MRC5 cells co-cultured with the irradiated eNOS overexpression cells than the vector cell line. Taken together, our results suggest that the induction of bystander effect can be promoted by the overexpression of NOS and the NO derived from the catalytic reaction of NOS after irradiation is the major factor in the induction of the bystander effects.Cited