Euthanasia of laboratory animals should be quick, painless and with a minimum of distress to the animal. Methods of euthanasia are the topic of an ongoing discussion, especially with respect to the degree of pain and distress associated with different methods. A common method of euthanasia of rats is intraperitoneal administration of pentobarbitone. Because of the alkaline character of this drug (pH≈10), it is thought to induce severe tissue irritation, and thus pain, of the viscera and peritoneum. A published study demonstrated a reduced number of neurones with c-fos like immunoreactivity, suggesting a reduced nociception, in the spine of rats receiving a mixture of pentobarbital and lidocaine, compared to rats receiving pentobarbital alone. However, it is our experience that visible signs of pain are not always observable during euthanasia. For this reason we performed a study comparing two concentrations of pentobarbitone (50 mg/ml vs. 200 mg/ml) for euthanasia in rats (n=12). The time point of loss of balance, immobility and respiratory stop were registered, and the behaviour was assessed from video recordings of the procedure. Our study revealed no differences between the two concentrations of pentobarbitone. Loss of balance occured after 70-73 seconds, immobility after 117-135 seconds, and respiratory arrest after 229-238 seconds. Moreover, the behaviour of the rats did not show overt sign of pain or distress. These findings are discussed.