In vitro susceptibility testing was done on urogenital isolates of Chlamydia trachomatis from five patients, four of whom were suspected treatment failures. At least one isolate from each patient was resistant to tetracycline at concentrations greater than or equal to micrograms/ml, although less than 1% of a population of organisms showed high-level resistance. Fully resistant populations selected by passage through 8 micrograms/ml tetracycline either died or lost their resistance on further passage in antibiotic-free medium. Relatively large inocula were required to demonstrate resistance, and morphology of inclusions was altered at high tetracycline concentrations. The observed resistance may be a new characteristic of the organism or merely newly recognized. Isolates resistant to tetracycline were resistant to doxycycline, erythromycin, sulfamethoxazole, and clindamycin but sensitive to rifampin, ciprofloxacin, and ofloxacin. Thus, resistance to tetracycline, erythromycin, and clindamycin occurs in C. trachomatis and may be a factor in some treatment failures.