The investigation of fetal pulmonary endothelial cell gene expression and function has been limited by the requirement for primary cells. In an effort to establish an immortalized cell line, ovine fetal pulmonary artery endothelial cells (PAECs;passage 5) were permanently transfected with the E6 and E7 open reading frames of human papillomavirus type 16, and phenotypes related to nitric oxide (NO) production were evaluated up to passage 28. Acetylated low-density lipoprotein uptake, endothelial NO synthase (eNOS) expression, and proliferation rates were unaltered by immortalization. Acetylcholine-stimulated eNOS activity was 218–255% above basal levels in immortalized cells, and this was comparable to the 250% increase seen in primary PAECs (passage 6). eNOS was also acutely activated by estradiol to levels 197–309% above basal, paralleling the stimulation obtained in primary cells. In addition, the expression of estrogen receptor-α, which has recently been shown to mediate the acute response in primary PAECs, was conserved. Thus fetal PAECs transfected with E6 and E7 show no signs of senescence with passage, and mechanisms of NO production, including those mediated by estradiol, are conserved. Immortalized PAECs will provide an excellent model for further studies of eNOS gene expression and function in fetal pulmonary endothelium.