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Use of self-administered vaginal swabs (SAS) for the detection of Chlamydia trachomatis by nucleic acid amplification tests simplifies specimen collection and transport, especially for women in nonclinical settings.We investigated the preference and comfort level of military women for the collection of SAS, compared with urine, for the diagnosis of genital chlamydial infections.During March through August 1999, female Army recruits in basic training at Fort Jackson, South Carolina, were invited to participate in the study. Participants were requested to complete a questionnaire after providing both first-void urine (FVU) and SAS specimens. Participant characteristics, preferences, and comfort levels were assessed using multivariate logistic regression.From 4496 eligible female recruits, 1403 (31%) completed questionnaires and 1382 provided both specimens; 11.8% (166 of 1403) of participants were infected with chlamydia. The relative sensitivity and specificity of the C. trachomatis Ligase Chain Reaction test on SAS in 1382 matched pairs was 81.1% and 98.6%, respectively, using the test result on urine specimens as the comparison standard. Most of the participants (90.8%) reported that they felt comfortable collecting the FVU specimen, and 69.6% indicated that they felt comfortable collecting SAS. Either specimen collection type received high acceptability at home and in the field, and more women reported that they would collect FVU than reported they would collect SAS in the future (in the field: FVU: 79.4%, SAS: 68.8%, P <0.001); at home: FVU: 90.9%, SAS: 82.9%, P <0.001). When questioned about ease of use, 60.4% of women reported that urine was the easier method. Preferences for SAS were associated with being white and having had sexual risk behaviors in the past 3 months.A study of preferences for urine versus self-administered vaginal swabs for the detection of C. trachomatis in military women showed that women generally found SAS acceptable. SAS should be a feasible alternative to urine collection in situations in which specimen storage or transport is an issue.