To investigate the influence of phenobarbital sodium on the action of morphine and on the diurnal rhythms of both opiate receptor binding and β-endorphin contents, the amount of specifically bound (³H)-morphine and immunoreactive β-endorphin were measured in the midbrain of phenobarbital-treated rats at 4h intervals in a day. Rats were housed and adapted to a controlled cycle of either 12 h light-12 h dark or 24 h constant dark. After 3 weeks of adaptation, 0.5 ml of physiological saline or phenobarbital sodium (20mg/kg/day, i.p.) were administered twice a day for 2 weeks. Highly significant diurnal rhythms of opiate receptor binding and β-endorphin were present in rat midbrain. In control group, the peak of maximum (³H)-morphine binding was observed at 22:00 h, whereas the peak of β-endorphin content was found at 06:00 h. Even in the absence of time cues these diurnal rhythms persisted, but they were highly modified with respect to the wave form as well as differences in the timing of peak and nadir. In the phenobarbital-treated group, these diurnal rhythms were also modified in shape, phase and amplitude, as well as in timing of peak and nadir. In this group, 24 h mean of opiate receptor binding was significantly decreased, while the 24 h mean level of β-endorphin content was highly increased. However, Kd values in all experimental groups did not change. This indicates that differences in binding were not due to changes in the affinity, but in the number of binding sites. Statistical analysis of regression line indicates that changes of receptor binding were closely correlated with the changes of β-endorphin content. These results suggest that phenobarbital may influence the action of morphine by changing the number of opiate receptors and that the modification of diurnal rhythm of opiate receptor by the agent is possibly due to changes of β-endorphin content.