Effects of morphine on the potentiation of pentobarbital-induced responses were investigated using μ-opioid receptor knockout mice. The duration of loss of righting reflex, hypothermia, and loss of motor coordination induced by pentobarbital were measured after pretreatment with either morphine or saline. Morphine pretreatment failed to show potentiation of both pentobarbital-induced loss of righting reflex and hypothermia in μ-opioid receptor knockout mice, while it significantly potentiated these responses in the wild-type controls. For motor incoordination test, morphine potentiated pentobarbital-induced motor incoordination in the wild-type mice. However, morphine may have opposite effects in the μ-opioid receptor knockout mice. These results demonstrate that synergism between morphine and pentobarbital is not detected in μ-opioid receptor knockout mice and that potentiation of pentobarbital-induced loss of righting reflex and hypothermia by morphine is mediated through μ-opioid receptor. It was interesting to note that pentobarbital-induced decrease in body temperature was less severe in μ-opioid receptor knockout mice than in wild-type mice.