We studied five different tests for diagnosing conjunctival Chlamydia trachomatis infection in specimens obtained from 100 children with moderate-to-severe trachoma. The tests were Giemsa stain, isolation in cell culture, direct fluorescein-conjugated monoclonal antibody, enzyme immunoassay, and a DNA probe. The Giemsa stain was least sensitive at 29%. The other tests gave essentially equivalent performances: sensitivity ranged from 73% to 84% and specificity from 93% to 100%. Seven of the 45 positive specimens were only positive in cell culture, whereas 11 of the 45 were negative in culture but positive in at least two nonculture tests. We speculate that these discordant results actually reflect the biologic status of the chlamydial infection. There appears to be a stage in the infection where chlamydial antigens and nucleic acids can be detected in the absence of infectivity. This stage of infection may contribute to the pathogenesis of trachoma by providing a source of sensitizing antigen.