Men with urethritis due to Neisseria gonorrhoeae were treated with gentamicin, which is inactive against Chlamydia. Urethral specimens were collected before treatment and one and two weeks after treatment and cultured for Chlamydia in irradiated McCoy cells. The overall incidence of chlamydial infection was 15 of 44 (34%). All of 15 Chlamydia-positive men and 11 of 29 Chlamydia-negative men (38%) developed postgonococcal urethritis two weeks after treatment. Pretreatment sera were examined by a complement fixation test and a simplified indirect fluorescent antibody (FA) test with a yolk sac-cultured antigen, strain SA2(f). Sera from all of 15 Chlamydia-positive men had titers of ⩾1:16 in the indirect FA test; 13 of 29 Chlamydia-negative men (45%) had positive tests. The complement fixation test was insensitive, detecting chlamydial antibodies in only one of 15 sera from Chlamydia-positive men. These results suggest that Chlamydia may cause many cases of postgonococcal urethritis.