Abstract <p><b>Objectives:</b> The ventral reticular formation of the medulla oblongata is an important inhibitory component of the pain control system. Glial cells and morphologically and physiologically heterogeneous populations of neuronal cells, the mediator specialization of which has not yet been precisely established, are involved in the regulation of pain signals. The aim of this study was to investigate the morphochemical organization of the ventral parts of the reticular formation of the medulla oblongata during the development of an acute pain reaction and diclofenac therapy. <b>Materials and methods:</b> A paw weight load scale was used to assess pain behavior. To assess motor activity, an open field test was performed 1 and 24 h after the injection of formalin. The study of the mediator profile of neuronal and glial cells in the reticular formation of the medulla oblongata was carried out using the immunoperoxidase reaction method to detect tyrosine hydroxylase (TH), neuronal form of NO synthase (nNOS), glial fibrillary acidic protein (GFAP) and the ionized calcium-binding adapter molecule 1 (iba-1). <b>Results and discussion:</b> In our study, acute pain caused an increase in the number of TH-positive neurons and astroglial activity in the ventrolateral reticular formation, while diclofenac reduced these indicators to control levels. The number of nNOS-positive neurons increased significantly with formalin-induced pain and diclofenac administration. Moreover, the motor activity of rats decreased 24 h after the injection of formalin, which was partially offset by analgesic activity of diclofenac. <b>Conclusion:</b> Acute pain caused by formalin injection resulted in increase in TH, nNOS and astroglial activity in the reticular formation of the medulla oblongata, while diclofenac showed moderate analgesic and anti-inflammatory effects in the condition.</p>