Two successive urethral swabs were used to obtain specimens for culture of Neisseria gonorrhoeae and Chlamydia trachomatis from 136 heterosexual men with urethritis. The first swab was used to culture N. gonorrhoeae and then C. trachomatis; the second was used to culture C. trachomatis only. C. trachomatis cultures from the second swab were positive more often (30 of 31 pairs) than were cultures from the first swab (22 of 31 pairs) (P less than .05). In addition, cultures from swab 2 had greater numbers of inclusions per coverslip more frequently (23 of 31 pairs) than did cultures from the first swab (six of 31 pairs) (P = .003). Numbers of chlamydial inclusions per coverslip were lower in specimens positive for both C. trachomatis and N. gonorrhoeae than in specimens positive for C. trachomatis only (P less than .02). In addition, the presence of N. gonorrhoeae in a specimen adversely affected the quality of the McCoy cell monolayer. In 17 of 21 instances of monolayer toxicity, cultures for N. gonorrhoeae were positive (P less than .01). These results demonstrate that when specimens from men with urethritis are cultured for N. gonorrhoeae and C. trachomatis, use of a second swab will improve rates of recovery of C. trachomatis. Material present in specimens that contain N. gonorrhoeae may adversely affect rates of isolation of C. trachomatis.