Slices of human corpora lutea of menstruation at different stages, or of inner and outer layers consisting of pure granulosa lutein cells and combined theca and granulosa lutein cells respectively, were incubated with [4-14C]-pregnenolone or with [4-14C]-androstenedione respectively. Conversion of 14C-radioactivity to progestins, androgens and/or estrogens was assessed by the reverse dilution technique with recrystallization to constant specific activity. Although progesterone was the major steroid formed from pregnenolone, a transient increase in androgen formation with a concomitant decrease in estrogen formation in developing corpora lutea was followed by an increase in estrogen with a decrease in androgen formation in mature corpora lutea. The reciprocal relation between estrogen and androgen formation is associated with the behaviour of theca lutein cells since the cells disappeared at the former and clearly differentiated at the latter stages of corpora lutea. The outer layers consistently produced more 17-hydroxyprogesterone, androgens and estrogens from pregnenolone than the inner layers, while the inner layers converted constantly more androstenedione to estrogens than the outer layers. It is suggested that aromatase activity of granulosa lutein cells in co-existence with theca lutein cells is required by human corpora lutea for fulfillment of the function of estrogen synthesis.