The present study was designed to observe the effects of acetylcholine (Ach) on the proliferative reaction of the splenocytes irradiated with low dose X-rays in Kunmin male mice in vivo and in vitro. The results showed that Ach enhanced significantly the spontaneous proliferations of the splenocytes and the responses of the splenocytes to ConA. But, the immune functional changes of the splenocytes irradiated with 75 mGy in vivo and in vitro were different. It would increase more in vivo, while less in vitro. These results suggest that Ach may stimulate the splenocyte proliferative reaction and participate in the immunoenhanced effect following low dose radiation to a certain extent. However, the action mechanism of Ach on the immune functional changes of the splenocytes irradiated with low dose X-rays in vivo and in vitro may be different. It is supposed to have relation to the change of the microenvironment of the splenocytes.