The antibiotic susceptibility of Chlamydia trachomatis isolates was determined in a tissue culture system. Representatives of all currently recognized serotypes of trachoma-inclusion conjunctivitis agents were tested. Tetracycline and erythromycin yielded similar results, with 1.0 μg/ml preventing chlamydial replication. Rifampin was the most active antibiotic, with 0.25 μg/ml completely suppressing inclusion formation of all strains. Fifty percent end points were usually achieved at one-fourth to one-eighth the 100% suppression level. Penicillin was not as effective, and the assays were often irregular. Antibiotic susceptibility of these chlamydiae was essentially the same, regardless of serotype, anatomic site infected, geographic origin, or antibiotic use in the community.