Previous studies have demonstrated an antinociceptive effect of brain-derived neurotrophic factor (BDNF) following infusion into the midbrain, near the periaqueductal grey and dorsal raphe nuclei. BDNF administration attenuated the behavioural response in the tail-flick and hot-plate tests, two models employing a phasic, thermal high-intensity nociceptive stimulus; the present studies extend our previous findings to include a model of moderate, continuous pain resulting from a chemical stimulus, the formalin test. Midbrain infusion of BDNF decreased the behavioural paw flinch response to subcutaneous formalin injection in both the early and late phases of the test. As our previous studies showed that BDNF-induced analgesia was reversible by naloxone, we have examined the effects of BDNF administration on brain and spinal cord levels of neuropeptides involved in the modulation of nociceptive information, including the endogenous opioid peptides, met-enkephalin and P-endorphin, as well as substance P and neuropeptide Y (NPY). At the site of infusion, within the PAG and dorsal raphe, BDNF increased the level of β-endorphin by 63%, but had no effect on substance P, metenkephalin or NPY levels. In the dorsal spinal cord, substance P (113% increase), β-endorphin (97% increase) and NPY (64% increase) were elevated, although ventral spinal cord levels of these peptides remained unchanged. These studies demonstrate a modulatory effect of BDNF on relevant neuropeptides within areas of the brain and spinal cord involved in the processing of nociceptive information.