Infections caused by Chlamydia spp are an important cause of human disease, and the accuracy and reproducibility of antimicrobial susceptibility tests for these bacteria could have considerable clinical implications. We have developed a reverse transcriptase PCR (RT-PCR) based method to determine the antibiotic susceptibility of Chlamydia spp., and compared this with conventional tests using immunofluoresence (IF) staining. The MICs of antimicrobial agents for a test strain of Chlamydia pneumoniae were higher by RT-PCR as compared with IF staining, indicating the greater stringency of the former method. Using RT-PCR, doxycycline and tetracycline were the most active agents (MIC 1 mg/L), followed by erythromycin (1.6 mg/L), and ciprofloxacin (16 mg/L). Neither trimethoprim nor sulphamethoxazole (400 mg/L) inhibited growth as assessed by both techniques. The RT-PCR based method may thus represent an improved and less time consuming assay for in-vitro determination of the antibiotic susceptibility of Chlamydia spp.