AIM:To study the effects of adrenocorticotropic hormone (ACTH)and electroacupuncture (EA)on formalin induced nitric oxide synthetase (NOS) positive neurons increases in the spinal cord of rats.METHODS:ACTH was administered by intrathecal injection (i.t.)and EA stimulation on“ jiaji” point was performed by 1 mA 50 Hz,5 mA 5 Hz and 1 mA 5 Hz respectively.The NOS positive neurons were assayed by NADPH diaphorase histochemistry.RESULTS:The results showed that both ACTH (0.5 u,i.t.)and EA stimulation (1 mA 50 Hz,5mA 5 Hz,lmA 5Hz)on “ jiaji” point 30 min significantly reduced the formalin induced NOS positive neurons in the rat dorsal horn.The combinative use of ACTH (0.5 u,i.t.)and EA(1 mA 5 Hz) caused a more marked reduction of the numbers of NOS positive neurons than that of the single ACTH or EA.Those effects were partially reversed by pretreatment with either the substrate of NOS,L arginine (10 nmol,i.t.)or opioid antagonist naloxone(10 g,i.t.).CONCLUSION:These results suggests that both ACTH and EA might inhibit the formalin induced NOS positive neurons increases and have a synergic effect acting via a different pathway.