From October 1973 through August 1974, 335 genitourinary tract specimens from patients with urethritis were inoculated into McCoy's cell cultures for the diagnosis of Chlamydia infections. Of the 45 Chlamydia isolates, 42 were recovered when glass vials rather than plastic microtiter plates were used as cell culture vessels. Herpes simplex virus was isolated 15 times. Bacterial overgrowth occurred 42 times; however, in 20 specimens the contamination was not apparent until after the first subculture. This is the first report of the frequency of Chlamydia infections in patients with urethritis in the Midwest and indicates that these organisms have a significant etiologic role in nongonococcal urethritis in this community.