A cell based assay for detection of endocrine disruptors has been previously developed using real time impedance monitoring of native endocrine signaling pathways in T47D breast cancer cell line.Stimulation of T47D cells with respective endocrine hormones (e.g.estrogen or progesterone)leads to distinct time dependent cellular changes (cell number change and/or cell morphological change) which can be detected by gold microelectrodes embedded in the bottom of the well of specialized microelectronic plates (EPlates).In the present study, a cell based co-cultivation assay was developed to study the complex signaling of steroid hormones including synthesis, secretion and paracrine signaling.In this assay, T47D cells expressing estrogen receptor (ER) as well as other steroid hormone receptors such as progesterone receptor (PR) were cultured on E-Plate and used as the responder cells;NCI-H295R cells (Human female adrenocortical cancer cell line) which express various steroid hormones (e.g.estrogen and progesterone) and steroidogenic enzymes (CYPs) was cultured in the inserts and used as effector cells.Co-culturing H295R cells with T47D cells induced a unique time-dependent biphasic response profile for T47D cells which correlated with increased T47D proliferation and morphological changes.The extent of the T47D response profile was dependent on the number of H295R cells co-cultured in the inserts.Using this assay, we have examined the effect of p450 enzyme inhibitor (Ketoconazole) and aromatase inhibitor (Anastrazole), both of which abolished the T47D response profiles exerted by the secreted hormones from NCI-H295R cells.This co-culture assay system makes it possible to identify compounds that may affect multiple aspects of endocrine signaling pathways including synthesis, secretion, receptor binding and induction of signaling pathways within the same assay.