Objective: To investigate the prevalence of ureaplasmas in semen and washed semen and to explore their effect on semen andrology variables. Design: Prospective study. Setting: In vitro fertilization (IVF) unit of a private hospital. Patient(s): Three hundred forty-three men participating in an assisted reproductive technology (ART) treatment cycle. Main outcome measure(s) The prevalence of ureaplasmas in semen and washed semen tested by culture, polymerase chain reaction assays, and indirect immunofluorescent antibody assays. Result(s): Ureaplasmas were detected in 73 of 343 (22%) semen samples and 29 of 343 (8.5%) washed semen samples. Ureaplasmas adherent to the surface of spermatozoa were demonstrated by indirect immunofluorescent antibody testing. Ureplasma parvum serovar 6 (36.6%) and U. urealyticum (30%) were the most prevalent isolates in washed semen. A comparison of the semen andrology variables of washed semen ureaplasma positive and negative groups demonstrated a lower proportion of nonmotile sperm in men ureaplasma positive for washed semen. Conclusion(s): Ureaplasmas are not always removed from semen by a standard ART washing procedure and can remain adherent to the surface of spermatozoa.