Urethral swab specimens collected from 108 male Japanese patients with acute nongonococcal urethritis (NGU) and from 50 Japanese men without NGU were examined for the presence of Mycoplasma fermentans, Mycoplasma penetrans, and Mycoplasma pirum by means of polymerase chain reaction-based assays. These mycoplasmas were not detected in any of the specimens, which suggests that they are unlikely to have a pathogenic role in acute NGU.