We evaluated the ability of the widely used spermicide nonoxynol-9, Conceptrol gel containing nonoxynol-9, and Conceptrol vehicle (without nonoxynol-9) to inhibit the formation of inclusions of Chlamydia trachomatis in cycloheximide-treated McCoy cells. Conceptrol vehicle produced a non-dose-related 40 to 59% reduction of the number of inclusions formed. In contrast, the addition of nonoxynol-9 and Conceptrol gel containing nonoxynol-9 at a concentration of 100 micrograms/ml reduced the number of inclusions by 84 to 95%. Prolongation of contact between nonoxynol-9 and the chlamydial organisms from 90 to 180 min did not result in additional inhibition. Using a higher concentration of chlamydial organisms and diluting the drug-chlamydia mixture before inoculation of the monolayers, we were able to test higher concentrations of nonoxynol-9. At concentrations of 100 to 5,000 micrograms of nonoxynol-9 per ml, a dose-related 19 to 84% reduction of the number of inclusions was noted.