Recent data suggest that ATP is released in the medulla in response to hypoxia and hypercapnia and may mediate excitation of presympathetic rostral ventro-lateral medulla (RVLM) neurones. Thus, during hypoxia or hypercapnia ATP is released within the areas where these neurones are located (Gourine et al. 2005b). Activation of P2 receptors in the RVLM evokes marked increases in blood pressure and renal sympathetic nerve activity (Horiuchi et al. 1999;Ralevic et al. 1999; Thomas et al. 2001) while exogenous ATP excites bulbospinal presympathetic RVLM neurones (Ralevic et al., 1999). However, further studies into the role of ATP in central respiratory and sympathetic chemosensitivity are hampered by the lack of selective pharmacological tools. The existing P2 antagonists (e.g. PPADS and others) are notorious for their lack of specificity and are not suitable for chronic in vivo studies. Here we validate a novel strategy to study the role of ATP-mediated signalling in the CNS based on a viral gene transfer of ATP-degrading enzymes. Extracellular ATP is broken down in several steps and this is catalyzed by ectonucleoside triphosphate diphosphohydrolases. Another family of enzymes, the alkaline phosphatases convert a variety of substrates, including ATP to adenosine. Here we have successfully used one such enzyme, the placental alkaline phosphatase (PLAP, the human gene) in a proof-of-principle experiment using a lentiviral vector LVV-EL1α-hPLAP. This LVV expresses PLAP under control of a non-specific EL1α promoter leading to the gene expression in neurones, glia and other cells. In rats (n=8) four unilateral injections of LVV-EL1α-hPLAP were made unilaterally into the right RVLM (under ketamine (60 mg/kg) and medetomidine (250 µg/kg) i.m. anaesthesia). Seven days later horizontal slices containing the ventral medullary surface were prepared and used for an in vitro experiments as described in (Gourine et al. 2005a; Gourine et al. 2005b). ATP microelectrode biosensors were used to determine release of ATP from the ventral surface chemosensitive areas in response to isohydric hypercapnia using aCSF solution in which NaHCO3 was increased to 50 mM (isosmotically replacing NaCl) and equilibrated with 10% CO2/90% O2 (pH ~ 7.45, pCO2 ~65 mmHg at 37°C). It was found that the amount of ATP released in response to isohydric hypercapnia was significantly (by ~50%; P=0.008) smaller on the transduced side of the medulla. Thus, expression of PLAP in the RVLM can be used as a highly effective approach for rapid degradation of ATP released into the extracellular space during chemosensory stimulation. We conclude that virally-mediated expression of ATP degrading ecto-enzymes can be used as a novel tool to study the multiple functional roles of ATP-mediated signalling in the central nervous system.