The endothelium plays a critical role in the growth and function of the developing pulmonary circulation. Studies of pulmonary endothelial cell gene expression and function have been limited by the requirement for early-passage primary cells due to senescence beyond passage 6-8. The purpose of the present study was to establish an immortalized fetal intrapulmonary artery endothelial cell line. As an example of a functionally-relevant phenotype, the conservation of acute endothelial nitric oxide synthase (eNOS) activation by estradiol was characterized by measuring 3H-L-arginine conversion to3 H-L-citrulline in intact cells. Ovine fetal intrapulmonary artery endothelial cells (PAEC) were sorted at passage 3 based on acetylated LDL uptake and transfected with a recombinant retroviral construct containing the E6 and E7 open reading frames of human papillomavirus type 16. These DNA viral genes inactivate host proteins involved in cell cycle control. Following selection, immortalized cells were propagated and studied at passages 18, 23, and 28, and compared to primary PAEC at passages 4-6. Acetylated LDL uptake and eNOS expression, indicative of an endothelial cell phenotype, were conserved in the immortalized cells. In addition, eNOS was acutely activated by 10-8 M estradiol in the immortalized cells, yielding NOS activity which was 346%, 279%, and 277% above basal at passage 18, 23, and 28, respectively. This level of activation was comparable to the 251% increase observed in primary cells. Acetylcholine (10-6M)-mediated eNOS activation was also demonstrable, yielding NOS activity that was 255%, 218%, and 285% above basal at passage 18, 23, and 28, respectively, compared to 221% in primary PAEC. The expression of estrogen receptor (ER) alpha, which mediates the acute response in primary PAEC, was also evident in the immortalized cells. These results indicate that fetal PAEC transfected with E6 and E7 show no signs of senescence with long-term passage, and that eNOS expression and ER-dependent acute activation of eNOS are conserved in these cells. The resulting cell line will provide an excellent model for further studies of eNOS regulation and other investigations of pulmonary endothelial cell gene expression and function.