Both clinical and epidemiological data suggest that inapparent infection byChlamydia trachomatisoccurs in humans. To confirm and study such infections, we developed a hybridization screening system directed toward chlamydial ribosomal RNA (rRNA). Six restriction endonuclease fragments derived from the clonedrrnAoperon of chlamydial serovar L2(434) were tested as hybridization screening probes, but only one fragment encoding the 5′ portion of the 16s rRNA gene plus some upstream flanking sequence was both sensitive and highly specific in such experiments. In Northern slot blot assays, hybridization analyses with this fragment as probe routinely detected one picogram or less of chlamydial RNA when that RNA was bound to membranes alone or as part of a mixture with a vast excess of mammalian RNA. The probe did not hybridize to RNA from mammalian and relevant bacterial sources but did hybridize to rRNA from B (ocular) and E (genital) serovars ofC. trachomatis.Experiments using RNA from conjunctival biopsies and standard conjunctival swab samples from cynomolgus monkeys showed that the probe reliably distinguishes between known chlamydia-infected and uninfected samples. This suggests that it may be useful for clinical screening. Characterization assays for the RNA-directed probe screening system in this monkey model of trachoma provide initial molecular evidence that ocular chlamydial infection may persist longer than previously thought, based solely on direct fluorescence antibody assay (DFA) and culture analyses.