It is well known that changes in CO2 concentration alter neuronal excitability. Whilst Hickman’s early attempts to promote CO2 anaesthesia met resistance (Antiquack, 1826) it is now routinely used on insects and small mammals. Despite this its mode of action remains obscure (Badre et al., 2005). Since CO2 reacts with water, catalyzed by carbonic anhydrase, there appear to be three possible activeagents: molecular CO2, pH and [HCO3-]. To distinguish between these we have combined CO2 with NH3. If the anaesthetic effect derives from the lipid soluble form of the gas (molecular CO2 or NH3) then the effect of the application of the two gases should be arithmetically additive. However, If the anaesthetic effect is due to acidosis produced by CO2 then NH3, which is well known to alkalinize, should be antagonistic. Finally, if the anaesthetic effect is due to [HCO3-] then NH3 should be synergistic with CO2 since NH4+ formation ‘mops-up’ H+ allowing a given CO2 concentration to produce more [HCO3-].We have used image analysis software to quantify the percentage of Drosophila remaining at the bottom of jars after being tapped-down following exposure to various gas concentrations. Within each experiment we averaged the response to 10 taps (30 s intervals). Fig 1A shows the percentage of Drosophila at the bottom of the jar 24 s after tap-down following exposure to NH3 for ~5 mins. Fig 1B shows data for CO2 exposure alone (CO2 KD50 ~60%) and following the addition of 0.1% NH3 on a CO2-background (CO2 KD50 ~20%). When applied alone both 40% O2 and 0.1% NH3 have little effect. However, adding 0.1% NH3 during CO2 exposure (CO2 >20%) produced a profound anaesthesia. These results are not consistent with either molecular CO2 or CO2-induced acidosis being the active agents. Indeed, they suggest a role for HCO3-. There are a number of mechanisms by which HCO3- may alter excitability. HCO3- may permeate GABAA receptors or it may act indirectly by altering Cl- or K+ equilibria. Finally, since pH buffering is proportional to [HCO3-] and [NH4+] we cannot rule out that these excitability changes occur through the modulation of physiological pH shifts which may drive vesicle-fusion (Caldwell et al., 2013). Unlike the other HCO3–dependent mechanisms the pH buffering hypothesis is also consistent with the sensitivity of vesicle fusion to the presence of other weak acids even in the absence of CO2 (Drapeau & Nachshen, 1988; Caldwell et al., 2013).