D-kyotorphin (D-Kyo) is a synthetic analogue of the neuropeptide kyotorphin and produces naloxone reversible analgesia. Stress-induced analgesia (SIA) is an in-built mammalian pain-suppression response that occurs during or following exposure to a stressful stimulus. The periaqueductal gray (PAG) is implicated as a critical site for processing strategies for coping with different types of stress and pain and NO affects its activity. The objectives of the present study were twofold: (1) to examine the effects of D-Kyo (5 mg/kg) on acute immobilization SIA; (2) to investigate the effect of peptide on NO activity in rat PAG after the stress procedure mentioned above. All drugs were injected intraperitoneally in male Wistar rats. The nociception was measured by the paw pressure and hot plate tests. A histochemical procedure for nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d)-reactive neurons was used as indirect marker of NO activity. Our results revealed that D-Kyo has modulating effects on acute immobilization stress-induced analgesia in rats may be by opioid and non-opioid systems. Although D-Kyo is incapable of crossing the blood-brain barrier it showed an increased number of NADPH-d reactive neurons in dorsolateral periaqueductal gray (dlPAG) in control but not in stressed groups. We may speculate that the effect of D-Kyo in the brain is due to structural and functional interaction between opioidergic and NO-ergic systems or D-Kyo appears itself as a stressor. Further studies are needed to clarify the exact mechanisms of its action.