Eight-week-old female NMRI-mice treated with 5 micrograms diethylstilbestrol (DES) or vehicle (olive oil) from d 1 to 5 after birth were hypophysectomized or sham operated. Thirty days after the operation, homogenates were prepared from the ovaries and incubated at 37 degrees C in the presence of [3H]pregnenolone for 1 h. In parallel experiments ovaries were taken for histology. In control ovaries regressing corpora lutea were still present 4 wk after hypophysectomy (Hx). Ovaries from DES females had no corpora lutea, but showed degeneration of the pre-Hx hypertrophic interstitial tissue. Steroids synthesized during the incubation were extracted and separated in a two-dimensional thin-layer chromatography system. After Hx, the amount of extractable [3H]testosterone, [3H]progesterone, and [3H]androstenedione recovered from homogenates of vehicle-treated females increased, but the amount of recovered [3H]-17 alpha-hydroxyprogesterone was not significantly altered. In contrast, in DES-treated females Hx significantly decreased levels of [3H]androstenedione and [3H]-17 alpha-hydroxyprogesterone, but did not affect [3H]progesterone or [3H]testosterone. The persistent difference in steroid synthetic activity after Hx could be linked to the difference in morphology between different ovarian tissue compartments of control and DES-treated females.