1. The aim of this study was to compare the behavioural and physiological responses to hanging and acute heat stress in three different chicken breeds. Chicks were obtained from a slow-growing French 'Label Rouge' line (SGL), a fast-growing standard line (FGL) and a heavy line (HL). The SGL, FGL and HL birds were slaughtered at their respective market ages of 12, 6 and 6 weeks, in an attempt to achieve similar body weights. Before stunning, birds were either shackled by their legs on the moving line for 2 min (shackling stress: SH) or placed in a room at 35 degrees C and 60% of humidity for 3.5 h and then shackled for 2 min (acute heat stress plus shackling: H + SH) or subjected to minimal stress by shackling for 10 s before stunning (control group: C). 2. Bird physiological responses to the three pre-slaughter treatments were estimated by measuring blood corticosterone, glycaemia, creatine kinase activity, acid-base status and electrolyte concentration as well as lactate content and glycolytic potential in the breast (Pectoralis major) and thigh (Ilio tibialis) muscles. Behavioural responses to shackling stress were evaluated by measuring wing flapping duration, straightening up attempts and vocalisations. 3. Blood corticosterone was higher in SH and H+SH groups than in the C group, regardless of genotype. The struggling activity on the shackle line differed among chicken breeds. It was more intense and occurred more rapidly after hanging in the SGL birds than in both other breeds. Furthermore, SGL struggling activity was not affected by hanging duration while it increased with hanging duration in FGL and HL birds. 4. Wing flapping duration was negatively correlated with blood pH, bicarbonate concentration and positively correlated with breast muscle lactate content, indicating that struggling stimulated antemortem glycolysis activity in breast muscle. Acute heat stress affected blood Ca2+ and Na+ concentration and increased glycaemia and glycolytic potential of thigh muscle. 5. Both acute heat stress and shackling before slaughter were experienced as stressful events by all types of birds.