Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are circulating hormones secreted predominantly in patients with hypertension or congestive heart failure. To obtain background data on plasma ANP and BNP levels in rats, we investigated the circadian rhythms and effects of anesthesia on these peptides. To determine the circadian rhythms, plasma samples from thirty rats were collected by non-anesthesia (decapitation) at six time points every fourth hour. To determine the effects of anesthesia, plasma samples from thirty-two rats were collected under diethyl ether, pentobarbital or urethane anesthesia. The plasma ANP and BNP levels were determined using a radioimmunoassay. The plasma ANP levels were high from the evening to early morning, while the plasma BNP levels were relatively low at 2:30 AM. The difference in the BNP levels was statistically significant. The plasma BNP levels were relatively high when the rats were anesthetized using urethane. These results suggest that blood collection should be performed between 10:30 AM to 2:30 PM to determine plasma ANP and BNP. The use of pentobarbital is also recommended for toxicological studies in rats.