A 1-year prospective study was undertaken of 65 prepubertal girls referred for evaluation of sexual abuse or vulvovaginitis to compare Chlamydiazyme, an enzyme immunoassay (EIA), with culture for the detection of Chlamydia trachomatis i in vaginal specimens. Five (8%) of 65 patients were initially EIA-positive. On repeat sampling without intervening antibiotic treatment, 4 girls who were initially positive became negative and 1 remained persistently positive. Chlamydial cultures were negative in all patients. The patient with the persistently positive EIA also had Group A streptococcal vaginitis. We were able to demonstrate that some strains of Group A Streptococcus can cause a positive reaction with the EIA. Cross-reactions with other bacterial species are also known to occur. We therefore conclude that Chlamydiazyme is not specific for the detection of chlamydial infection in the vagina of prepubertal girls and, for medicolegal reasons, should not be used for evaluation of sexual abuse.