By means of Fos immunocytochemistry, nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry and microinjection methods, the role of nitric oxide synthase (NOS) of dorsal raphe (DR) neurons in the modulation of rats sigmoid pain was studied. The results showed: (1) Rats exhibited aversive behavioral responses related to visceral pain after injecting formalin into the sigmoid wall. NOS neurons in DR were up-regulated, in addition, about 8% of NOS-labeled neurons were Fos positive. By contrast, there were no Fos/NOS double-labeled neurons in the control group. (2) Formalin-induced sigmoid pain scores and the expression of Fos in the spinal cord at S1 segment were decreased after microinjecting L-NAME into the DR. These findings suggest that NOS neurons are involved in the modulation of formalin-induced sigmoid pain and that NO may play an important role in the transmission of visceral nociceptive message in the midbrain.