Treatment of HeLa 229 cultures with 17-beta-estradiol or with diethylstilbestrol, a synthetic estrogen analog, prior to infection with Chlamydia trachomatis UW31 (serovar K) or LGV440 (serovar L1) led to a 50 to 60% enhancement of chlamydial inclusion formation. After infection, the presence of estrogen was required for the enhancement. The optimal concentration of estrogen required was 10(-10) M. At least 18 h of preinfection treatment plus 12 h of postinfection treatment was necessary. The adherence of purified radioactive elementary bodies of C. trachomatis to estrogen-treated HeLa cells was stimulated in an estrogen dose- and exposure-dependent manner. The requirements for both pre- and postinfection exposure to the hormone suggest that alterations in the cell membrane as well as in the metabolic capacity of the host cells is required for intracellular chlamydial development. Cycloheximide did not prevent estrogen enhancement of chlamydial adherence or subsequent intracellular development of inclusions.