Disease outbreaks can require the rapid, humane culling of large numbers of animals, preferably with minimal human health risk in the case of zoonoses. One technique for poultry is to kill them in situ, by injecting liquid carbon dioxide into the house. However, because of the low temperature (-78 oC) of liquid CO2, birds might suffer from hypothermia prior to unconsciousness. This study investigated the behaviour of hens during liquid CO2 injection while monitoring CO2 levels and house temperature. A 3-tier conventional cage unit housing approximately 28,000 hens was fitted with a lance injection system that injected liquid CO2 from a tanker into the deep pit. Seven cages were fitted with temperature loggers, three cages with CO2 sampling tubes (fitted to a CO2 analyser), and four infrared CCTV cameras (to monitor behaviour) were mounted above top tiers of cages. Side inlet vents and the deep-pit were sealed with plastic prior to gassing. The ambient temperature rose to 27 oC just prior to CO2 injection. Approximately 1.22 kg/m3 of CO2 was injected into the house over 27.5 min, to reach a concentration of 60%. Respiratory distress (gasping) began 147±22 (mean±SD) sec after CO2 application (mean CO2=0.7±0.8%). Loss of balance and convulsions, indicative of birds losing consciousness, were observed at 215±18 and 276±14 sec (respectively) after CO2 application, when corresponding CO2 levels were 3.5±3.0% and 6.0±4.6% and when the house temperature was still above 25 oC. Visible birds ceased moving 731±47 sec (i.e.~12 min) after CO2 application, which was before the temperature had fallen below 20 oC and mean CO2 levels were 27.3±6.8%. Data indicate that birds die before they suffer from hypothermia. Although CO2 can cause respiratory distress, birds begin to lose consciousness in ~4 min, which is quicker than catching and culling, and safer for human operators.