Detection of chlamydial infections depends on the sensitivity of the techniques used. Variables include the number of body sites sampled, the number of samples obtained, and the number of passages in tissue culture. To assess these factors, microdilution plate cultures with a single blind passage were performed on specimens from 10,291 men and women attending a sexually transmitted disease clinic. Overall, 21% of the men and 30% of the women were culture positive. However, 18% of endocervical, 28% of female urethral, and 29% of male urethral cultures that were positive became so only after a single passage. Of culture-positive women, 23% were positive at the urethra only. Pooled urethral and endocervical specimens were positive more often than an endocervical specimen alone but less often than separately cultured endocervical and urethral specimens. A total of 221 specimens from 92 men and 66 women were subjected to five serial blind passages. Of 83 positive specimens, 29 (35%) were positive only after two or more passages. A total of 37 (46%) women were culture-positive, but only 12 (33%) of those who were positive and had an endocervical culture would have been detected by a single endocervical culture that was not passaged. The sensitivity of chlamydial culture is substantially less than 100% but can be improved by culturing samples from both the urethra and endocervix in women and by serial passage in tissue culture.