The bystander effect on unirradiated neoplastic human salivary gland (HSGc) cells was investigated by co-culturing them with HSGc cells that had been irradiated with 290 MeV/u carbon beams of different linear energy transfer (LET) values. It was found that the plating efficiency and proliferation of the unirradiated recipient cells were increased and that these increases were related to the LET as well as the radiation dose. Exposure of HSGc cells to higher LET and higher dose was much more effective in enhancing the plating efficiency and proliferation of the unirradiated cells than exposure to lower LET and lower dose. However, when PTIO, a nitric oxide (NO)-specific scavenger, was present in the coculture medium, the cell growth capacity of the unirradiated recipients was reduced to control level, indicating that NO is involved in the bystander response. As an oxidization product of NO, nitrite was detected in the coculture medium and its concentration depended on the LET and dose of irradiation. Using a NO-generator sper/ NO, it was verified that NO at low concentrations indeed enhanced cell proliferation. Accordingly, NO plays an important role in medium-mediated bystander effects.