The radiation-induced bystander response is defined as a response in cells that have not been directly targeted by radiation but that are in the neighborhood of cells that have been directly exposed. In the work described here, it is shown that bystander cell killing of normal human fibroblast WI-38 cells was induced by synchrotron microbeam X radiation. Cell nuclei in confluent WI-38 cells were irradiated with the microbeam. All of the cells on the dish were harvested and plated 24 h after irradiation. It was found that the bystander cell killing effect showed a parabolic relationship to the radiation dose when five cells were irradiated. At doses above 1.9 Gy, the surviving fraction increased to approximately 1.0. This suggests that induction of bystander cell killing may require some type of activity in the targeted cells, because the dose resulting in 37% cell survival was about 2.0 Gy. Bystander cell killing was suppressed by a pretreatment with aminoguanidine [an inhibitor of inducible nitric oxide (NO) synthase] or carboxy-PTIO (a scavenger of NO). These results suggest that NO is the chief initiator/mediator of bystander cell killing induced by X-ray microbeams.