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To compare laboratory to laboratory variability in methods of cell culture for Chlamydia trachomatis performed by North American research laboratories.The authors administered a standardized 54-question survey to laboratories that had published articles in any of three medical journals reporting on the use of cell culture to identify individuals with C. trachomatis infection. Laboratory to laboratory variability in specimen collection, specimen transport conditions, culture methodologies, and criteria for evaluation of culture outcomes was examined.Twenty-five (93%) of 27 laboratories responded to the survey. Only two of 54 questions were answered uniformly by all responding laboratories. All laboratories reported vortexing or sonication of specimens before culture inoculation and centrifugation of inoculated cultures prior to incubation. In contrast, substantial variation was noted in specimen collection devices, specimen transport conditions and times, culture format, culture procedures, and criteria for identifying positive cultures.Although this study did not evaluate the sensitivity of chlamydia cell cultures performed in different laboratories, there was substantial laboratory to laboratory variation in nearly every facet of culture evaluated. Laboratory to laboratory variation in chlamydia cell culture sensitivity likely accounts for part of the substantial variability in published evaluations of the sensitivity of nonculture chlamydia diagnostic tests.