Nonoxynol-9, a nonionic detergent and active ingredient in spermicidal contraceptives, has been reported to have anti-chlamydial properties. However, in this study exposure of elementary bodies of Chlamydia trachomatis serovar E to nonoxynol-9 (12.5-10,000 micrograms/ml) had no effect on chlamydial infectivity. In contrast, uninfected McCoy cells incubated with increasing concentrations of nonoxynol-9 over 72 h displayed dose-related cytotoxicity. When infected McCoy cells were exposed to nonoxynol-9, the developing chlamydial inclusions did not stain with iodine even though they were similar in number and appearance to the inclusions in unexposed, infected monolayers. Transmission electron microscopy of nonoxynol-treated, infected cells revealed apparent damage to the inclusion membrane and reticulate bodies within. The infectivity of the chlamydiae in the iodine-negative inclusions on subpassage was only 0.3%. We conclude that the primary action of nonoxynol-9 is on the McCoy cell and that there may be secondary effects on the intracellular parasite.