Publisher Summary This chapter discusses the generation and application of ovarian steroidogenic cell lines. Primary granulosa cells of mammals can be immortalized by transfecting them with oncogenes and mutated tumor suppressor genes. The established cell lines preserve their potential to undergo differentiation and luteinization under stimulation by substances elevating intracellular levels of cyclic adenosine monophosphate (cAMP). Moreover, if these cells are cotransfected with plasmids coding for the rat or human luteinizing hormone (LH), human chorionic gonadotropin (hCG), or follicle-stimulating hormone (FSH) receptors, they respond to gonadotropin stimulation by activation of hormone-sensitive adenylate cyclase followed by de novo formation of steroid acute regulatory (StAR) protein; the cytochrome P450 side chain cleavage enzyme system; and subsequent formation of pregnenolone, progesterone, and 20α adihydroprogesterone. These cells also respond to gonadotropin/cAMP stimulation, elevating intracellular communication via gap junctions (GJs) and forming typical ovarian paracrine factors such as follistatin. The advantage of this cell system is its homogenous population of cells because each cell line is monoclonal. Thus endocrinological, biochemical, and molecular studies on the entire cell population can be applied to an individual cell type.