A long-term effect of pentobarbital treatment on the storage and release of atrial natriuretic peptide (ANP) was investigated in rats. The experimental group was treated with pentobarbital (50 mg/kg, ip), and the control group was injected with the vehicle only. They were used one week after the treatment. Male and female rats were used separately to see if there exists a sex difference in the response to pentobarbital. In male rats, the plasma ANP measured in a conscious state was significantly higher in the experimental group than in the control. The ANP content as well as the number of specific granules in the atrial tissue was significantly lower in the experimental group. In response to extracellular volume expansion (VE), amounting up to 5% of the body weight over 30 min, while the plasma ANP increased in the control, it did not significantly change in the experimental group. Urinary responses to the exogenous infusion of ANP did not differ in magnitude between the two groups. In female rats, neither the plasma level nor the atrial content of ANP (or the atrial granularity) was different between the experimental and control groups. Nor did the increase of plasma ANP in response to VE differ in magnitude between the two groups in female rats. These results indicate that pentobarbital may have a long-term effect on the storage and release of ANP.