The authors have shown that protein antigens, RNA, and DNA from Chlamydia trachomatis are present in synovial tissues of patients with Reiter's syndrome (RS). However, those studies gave no insight into the host cell type involved or the precise tissue location of the bacteria. To address such issues, the authors developed an in situ hybridization system to detect chlamydia, and they used that system to examine synovial biopsies from a patient with RS and a patient without RS. The in situ system uses a previously described digoxigenin-labeled DNA probe that hybridizes with chlamydial 16S rRNA sequences in paraformaldehyde-fixed samples. Control studies with chlamydia-infected and uninfected HeLa cells confirmed that the in situ system is as sensitive as is direct fluorescence cytology for detection of the organism. Morphology of host and chlamydia cells is preserved after hybridization. Studies using synovial tissue from an osteoarthritis patient produced no in situ hybridization signal, but similar hybridization to tissue from a culture-/direct fluorescence cytology- negative RS patient had a strong intracellular signal for chlamydia within a subsynovial cell layer. These in situ hybridization results confirm the extensive presence of chlamydia in synovia and extend the authors' earlier observation that chlamydia RNA is present in the synovia of patients with RS. The data also confirm their electron microscopy studies, indicating that chlamydia are intracellular in synovial tissue, and they further show that infected host cells are located beneath the synovial lining.