The present study examined a long-term effect of pentobarbital anesthesia on the granularity of atrial cardiocyte in rats. The male and female rats were used separately and the animals were divided into experimental and control groups. The experimental group of rats were treated with pentobarbital (50mg/kg, ip) and were sacrificed one week after the treatment. The cardiocytes of the right atrium were examined with electron microscopy and the number of atrial granules, atrial natriuretic peptide (ANP) granules, was calculated. The atrial cardiocytes showed similar morphology between experimental and control animals in both sexes except the number of ANP granules. The cardiocytes contained well developed Golgi apparatus, ribosomes, and ANP granules near the nuclear poles. The ANP granules, 0.2-0.4 micrometer in diameter, were round in shape, delimited by a distinct membrane, and exhibited a homogeneous dense matrix. The cytoplasm near the sarcolemma had some vesicles. In male rats the number of ANP granules in atrial cardiocytes of the experimental group significantly decreased (p<0.01) than that of the control group. In female rats, however, the number of ANP granules did not significantly differ between the experimental and control groups. These results suggest that pentobarbital has a long-term decrease in ANP granules of atrial cardiocytes in male rats, however, the mechanism of which is still unknown.