The objective of this study was to examine the effect of an anaesthesia using 70% carbon dioxide and 30% oxygen on endocrine stress reaction, behaviour and clinical parameters of male suckling piglets during castration. One hundred and seventy one male piglets, three to five days of age, were allocated to two experiments. They were assigned either to the procedures control handling, control castration, handling under anaesthesia or castration under anaesthesia in each experiment. In Experiment 1, adrenaline and noradrenaline plasma concentrations were measured in blood samples taken before (-15 min) and after (immediately, 2 min) handling/castration. In Experiment 2, behavioural observations and clinical parameters such as heart and respiratory rate, oxygen saturation, reflexes and recovery time were assessed at several sampling times. Measurement of adrenaline and noradrenaline concentrations revealed an increase in all groups after handling/castration (p < 0.0167), but higher concentrations were seen in the anaesthetized groups (25 to 93 times) than in control groups (two to four times). The excessive endocrine reaction suggests that carbon dioxide inhalation causes a more stressful situation in piglets compared to castration without anaesthesia. Behavioural abnormalities, significant decreases in the heart rate, the respiratory rate and the oxygen saturation (p < or = 0.001) including a cardiac arrhythmia (extrasystoles) underline the impression that CO2 inhalation anaesthesia negatively affects animal welfare. Based on the results of this study, this anaesthetic method is unsuitable to reduce stress induced by castration. Further research on alternatives is necessary to ensure the well-being of the piglets during castration.