In human amnion-derived WISH cells [³H]estradiol-17β binding sites are not detectable, but they become measurable in cells exposed to cAMP elevating agents such as forskolin or Ro 20-1724. In cells unexposed to these drugs, 17β-estradiol stimulates prostaglandin (PG)E₂ release but exerts an evident inhibitory effect in cells exposed to Ro 20-1724. Both stimulatory and inhibitory actions are inhibited by the estrogen receptor antagonist, tamoxifen, by cell pretreatment with cycloheximide, or when the hormone is bound to BSA. Our data demonstrate for the first time that 1) 17β-estradiol modulates PGE₂ release from WISH cells, interacting with specific intracellular receptors and probably evoking new protein synthesis, and 2) WISH cell responsiveness to 17β-estradiol seems to be modulated by cAMP, whose levels are significantly increased by the steroid hormone in the presence of Ro 20-1724. The nucleotide is presumably responsible for the enhacement of hormone receptor availability and for the inhibition of PGE₂ release observed in the presence of Ro 20-1724.