The antibacterial, antifungal and antimycoplasmal activity of human semen was studied. Gram-positive aerobic bacterial species i.e. staphylococci, but not gram-negative aerobic bacteria, were inhibited by seminal fluid in vitro. Neither were anaerobic gram-positive or gram-negative bacteria, nor Candida or Mycoplasma inhibited. Semen of healthy males had a higher antibacterial effect on S. albus than that of patients with symptoms of chronic prostatitis. There was a positive correlation between the antibacterial power of the semen of the patients studied and their content of zinc and magnesium, while no correlation was found with fructose and lysozyme or the number of spermatozoa in any of the groups. A positive correlation was found between the antibacterial capacity and the volume of the ejaculate in the patients but not among the controls. The antibacterial substance(s) was dialysable, ether-extractable, resistant to boiling and partly to storage at room temperature. The addition of EDTA, tranexamic acid and ammonium reineckate to semen did not influence the antibacterial effect, which was, however, slightly inhibited by sodium polyanethol sulphonate. The nature of possible antibacterial substances in semen is discussed.