In a prospective study, the prevalence of infection with Chlamydia trachomatis and Ureaplasma urealyticum was evaluated in the semen of 92 asymptomatic male partners of infertile couples using polymerase chain reaction and culture, respectively. The results were compared with the detection of serologically specific antibodies. U. urealyticum and C. trachomatis were detected in 12 (13%) and 10 (10.8%) of the tested ejaculates, respectively. One mixed infection was detected. No correlation was found between detection of the pathogens in ejaculates and the presence of specific antibodies in serum. This study therefore confirms the limited diagnostic value of serological analysis to ascertain infection with C. trachomatis or U. urealyticum. The high frequency of detection of these pathogens among asymptomatic male partners of infertile couples emphasizes their potential role in the impairment of male fertility, and the need for sensitive and specific detection methods to prevent infection of the early embryo when using new reproductive techniques such as zona pellucida hatching or intracytoplasmic microinjection.