The antinociceptive action of botulinum toxin type A (BT-A) has been the subject of preclinical and clinical research for more than 10 years. Until now, various experimental models of inflammatory and neuropathic pain have shown long-term antinociceptive activity of BT-A after its single local administration, which differentiates it from all known analgesics. It has been shown that BT-A reduces formalin-induced pain, as well as expression of c-Fos in the dorsal horn of the spinal cord (the first synapse in the transmission of painful information), if administered peripherally (in paw pad), but also intrathecally (in the spinal canal). However, there is no data on the possible effect of BT-A on peripherally caused pain in higher areas of the nociceptive pathway. Therefore, the aim of this study was to investigate the effect of BT-A on the expression of c-Fos protein in the periaqueductal gray matter (PAG), one of the regions of the brain that plays an important role in the modulation of nociception. Immunohistochemistry was used to investigate the immunoreactivity of c-Fos positive neurons in animal brain tissue samples after inducing inflammatory pain by peripheral 5% formalin injection. One group of animals was pre-treated with BT-A injected into the lateral cerebral ventricles (i.c.v.) at a dose of 1 i.j./kg while in the control group i.c.v. saline was injected in the same way. The results show that peripheral injection of formalin causes activation of neurons in PAG, as expected based on results from previous studies. However, after i.c.v. administration to BT-A does not reduce the activation of neurons in that brain region. These results support the hypothesis of segmental activity of BT-A on the pain of peripheral origin.