OBJECTIVE To clarify the effect of androgen concentration on blood flow regulation in seminal vesicles and to identify the androgen most responsible for this effect. METHODS The androgen concentrations in the seminal vesicles and the seminal vesicle blood flow were measured at 0, 3, 6, 12, 24, 48, and 72 hours after castration. The androgen concentration was measured with a newly developed highly sensitive simultaneous androgen quantification method that uses liquid chromatography-tandem mass spectrometry. The blood flow was measured with the radioactive microsphere injection method. The change in seminal vesicle blood flow in 6-hour castrated rats after administration of 3 mg/kg testosterone, 3 mg/kg dihydrotestosterone (DHT), 3 mg/kg dehydroepiandrosterone, or 3 mg/kg testosterone plus 20 mg/kg finasteride was evaluated. RESULTS A correlation was observed between the DHT concentration and blood flow in the rat seminal vesicle after castration. The DHT concentration and blood flow decreased after castration to 31.6% and 37.9%, respectively, of the normal level at 6 hours. The decline in DHT concentration and blood flow decreased further thereafter to 2.2% and 18.1%, respectively, of the normal level at 72 hours. Both testosterone and DHT fully restored the organ blood flow in castrated rat seminal vesicles. However, the blood-flow-increasing effect of testosterone was attenuated by more than 50% when administered in combination with finasteride. Dehydroepiandrosterone did not increase seminal vesicle blood flow. CONCLUSIONS The results of our study showed a strong correlation between the organ DHT concentration and blood flow in rat seminal vesicles. Of the various androgens, the most active androgen in the regulation of seminal vesicle blood flow was DHT.