The aims of this study were: first, to investigate the effects of anaesthesia on phosphorylated extracellular signal-regulated kinase (p-ERK)1/2-immunoreactivity (-ir) in the brainstem; second, to choose the best anaesthetic for p-ERK1/2 studies; and third, to determine the effect of short-term hypotension on p-ERK1/2-ir in the brainstem. Rats were anaesthetized with halothane, sodium pentobarbital or 100% CO2 narcosis, or were cervically dislocated and within 5 min perfused and the brains processed immunohistochemically for pERK1/2-ir. p-ERK1/2-ir was primarily observed in regions associated with cardiovascular and/or respiratory control. Several regions consistently showed dense p-ERK1/2 labelling, including a restricted region of the ventrolateral medulla (VLM). In contrast, other regions showed differential labelling depending on the mode of death. Cervical dislocation showed the least VLM labelling, limited to a discrete area approximately 0.6-1.4 mm caudal to the facial nucleus. Anaesthetics induced labelling throughout the VLM, with halothane inducing the most. Many p-ERK1/2-ir VLM neurons were catecholaminergic following halothane or sodium pentobarbital anaesthesia, but no double labelling was seen following cervical dislocation. Of the anaesthetics, sodium pentobarbital induced the least labelling and was used subsequently. Intravenous hydralazine was used to induce a 20-min period of hypotension, whereas arterial pressure did not change in vehicle-treated animals. Hydralazine evoked more pERK-ir neurons in specific regions, including the VLM, nucleus tractus solitarius (NTS), parabrachial nuclei, Kolliker-Fuse nucleus and locus coeruleus. Approximately twice as many p-ERK1/2-positive neurons were seen in the intermediate NTS and rostral VLM following hydralazine compared with the vehicle. In conclusion, p-ERK1/2-ir identifies neurons in central autonomic regions, and their number and distribution are markedly affected by anaesthetics, and are increased in some regions by short-term hypotension.