A simple and sensitive microtiter plate estrogen bioassay based on stimulation of alkaline phosphatase in Ishikawa cells: estrogenic action of delta 5 adrenal steroids. We have developed an estrogen bioassay using the Ishikawa human endometrial adenocarcinoma cell line growing in 96-well microtiter plates. Alkaline phosphatase enzyme activity (AlkP) in these cells is markedly stimulated by estrogens, and this enzyme can be easily quantified in situ using a chromogenic substrate. These cells are very sensitive to estrogens; estradiol induces AlkP at levels as low as 10(-12) M. Antiestrogens completely block the action of estradiol. Various estrogens stimulate AlkP with potencies comparable to those achieved in vivo. The induction of AlkP is specific for estrogens; no other type of steroid, including androgens, progestins, mineralocorticoids, or glucocorticoids produce this effect. The stimulation of AlkP in Ishikawa cells is specific for estrogens, is highly reproducible and sensitive, and permits large numbers of samples to be assayed with ease. We have used this assay to investigate the estrogenic action of the adrenal delta 5-3 beta-hydroxysteroids. While pregnenolone is inactive, dehydroepiandrosterone and its sulfate ester induce AlkP slightly. However, the C19 steroid, 5-androstene-3 beta, 17 beta-diol is considerably more estrogenic in this assay, although it stimulates Ishikawa AlkP with a potency of 1/30,000 that of estradiol. The stimulation by 5-androstene-3 beta,17 beta-diol is inhibited by antiestrogens, but it is not blocked by the delta 5-3 beta-hydroxysteroid isomerase/dehydrogenase inhibitor, cyanoketone, or by the aromatase inhibitor, 4-hydroxy-androstenedione. Thus, neither conversion to a delta 4-3-ketone nor aromatization is required for the action of this unusual estrogen. 