The antimicrobial susceptibility of a low-laboratory-passage, slow-growing, genital Chlamydia trachomatis strain was studied by five different procedures with the use of McCoy cells pretreated with 5-iodo-2-deoxyuridine. The effects of antimicrobial agents when added to cultures on day 0 or day 2 after inoculation with C. trachomatis and the effects of washing and reincubating treated cultures in antimicrobial-free media were investigated. Tetracycline and erythromycin inhibited C. trachomatis growth at concentrations attainable in human serum, although their actions were reversible and significantly higher concentrations were needed to "cure" 48-h infected cultures. On a weight basis, spectinomycin was relatively ineffective in inhibiting C. trachomatis growth. The minimal inhibitory concentration of penicillin measured by our assay procedures was higher than that reported by other investigators. The five assay procedures used in this study were reproducible, and our results indicate that we can obtain more pertinent information about the efficacy of an antimicrobial agent in controlling C. trachomatis growth by using a combination of these assays than by simple minimal inhibitory concentration determinations, as had been previously described by other investigators. In addition, we failed to demonstrate changes in tetracycline susceptibility of C. trachomatis isolates from two patients who had received tetracycline therapy.