OBJECTIVE Whether sperm normally reside in the seminal vesicles of fertile men without ejaculatory duct obstruction, and the effect of duration of sexual abstinence on results of seminal vesicle aspiration were determined. METHODS Bilateral seminal vesicle aspiration was performed on 12 fertile volunteers under transrectal ultrasound guidance with randomization according to 0 and 5 days of abstinence. Seminal vesicle aspirates were examined microscopically for number and motility of sperm. A positive aspirate was defined as greater than 3 sperm per high power microscopic field. RESULTS Duration of abstinence had a significant effect on outcome of seminal vesicle aspiration in fertile volunteers. No volunteer with 0 days of abstinence had a positive aspirate from either seminal vesicle, whereas a third of those with 5 days of abstinence had at least 1 positive seminal vesicle aspirate. CONCLUSIONS Significant numbers of sperm are not normally found in the seminal vesicles of fertile men immediately after ejaculation. Diagnostic seminal vesicle aspiration should be performed after 0 days of abstinence. Longer periods of sexual abstinence may be used in patients undergoing seminal vesicle harvesting of sperm for assisted reproduction.