Ovarian granulosa cells are the primary site of estrogen and progesterone synthesis and play an essential role in the maturation of the developing ovum. Freshly isolated granulosa cells are often used to study the regulation of steroid and protein biosynthesis, but the small number of cells available for these cultures has proven inadequate for many detailed gene regulatory studies. The goal of this study was to develop human granulosa (HG) cell lines that maintain differentiated function. The E6 and E7 open reading frames of high risk strains of human papillomavirus have been used to produce immortalized cell lines. Primary cultures of human luteinized granulosa cells were infected with defective retroviruses containing the E6 and E7 regions of human papillomavirus 16 and with the neomycin phosphotransferase gene to confer G418 resistance. Three of eight clones that were isolated after selection in medium containing G418 were found to produce progesterone following treatment with forskolin or dibutyryl cAMP for 48 h. Forskolin caused these cells to retract in the characteristic rounding response, as described in primary HG cultures. One clone, HGL5, was used for a detailed characterization of differentiated function. HGL5 cells retained the ability to increase progesterone production and convert exogenously added androstenedione to estradiol in response to agonists of the protein kinase-A pathway (forskolin and dibutyryl cAMP), but were not responsive to FSH or LH treatment. A key enzyme in the production of estradiol, cytochrome P450 aromatase, has proven difficult to maintain in long term cultures of granulosa cells. For that reason, we examined the expression of aromatase in the transformed HGL5 clone by monitoring mRNA levels. Aromatase mRNA increased by 4- to 5-fold after forskolin treatment, as determined by Northern analysis. This human granulosa cell culture line maintains many of the functions of normal cells and should provide an important model to study the molecular events controlling granulosa cell differentiation and function.