The clinical significance of microorganisms in semen is unclear, as they may signify contamination rather than infection. Specimens from six locations in 97 healthy men scheduled for vasectomy were analyzed. Totally 1033 strains, aerobic or anaerobic, were isolated. Of 61 intraoperatively obtained vas deferens cultures, only one was positive. Of the 97 semen samples, 83% contained bacteria, 140 aerobes and 113 anaerobes; 44% of the strains found in semen and 58% of those in prostatic secretion were also identified in the urethra. The finding that 71% of the strains colonizing the coronal sulcus were present in the urethra indicates that the distal part of the urethra is colonized by a bacterial flora similar to that in the sulcus. The urethral flora may then contaminate semen and prostatic secretion. Presence of multiple bacterial species in semen was not associated with abnormal sperm function.