The in vitro activity of nonoxynol-9 against four serotypes (C, D, H, and K) of Chlamydia trachomatis was investigated. A constant inoculum of each serotype was exposed to serial twofold dilutions (1:100 to 1:800) of Koromex, Conceptrol, or reference preparations (not containing nonoxynol-9) for 4 and 24 h at 37 degrees C. The mixtures of nonoxynal-9 or nonnonoxynol preparations and control inocula were dispensed into triplicate wells containing McCoy cell monolayers. After incubation at 37 degrees C, the monolayers were fixed and stained with iodine and examined for evidence of infection with C. trachomatis. All nonoxynol-9-containing preparations showed marked antichlamydial activity as judged by percent reduction of glycogen-containing intracytoplasmic inclusions. The reference preparations, which did not contain nonoxynol-9, were markedly less active when tested in this in vitro system.