Increased 5-lipoxygenase (5LO) expression in pulmonary artery endothelial cells (PAECs) has been observed in primary pulmonary hypertension, a disorder associated with pulmonary vascular remodeling and aberrant endothelial cell proliferation. To examine whether 5LO plays a role in endothelial cell proliferation, we analyzed the effect of 5LO inhibitors on cultured human PAECs. Analysis of [(3)H]thymidine incorporation showed that 5LO and 5LO-activating protein inhibitors AA-861, nordihydroguaiaretic acid (NDGA), and MK-886 all inhibited PAEC growth in a dose-dependent manner, with maximal inhibition of >90% and IC(50) values of 3.9, 1.8, and 0.48 microM, respectively. The effect of AA-861 and NDGA correlated with their effect on 5LO activity in PAECs. Concentrations of these inhibitors at or below their IC(90) values did not cause significant cell death as determined by lactate dehydrogenase release, but decreased cell doubling, as measured by cell counting at 24 h after serum replenishment. Analysis of DNA content suggested that the inhibitors led to an accumulation of PAECs at the G(0)/G(1) phase. Antisense oligonucleotides to 5LO mRNA delivered at a transfection efficiency of approximately 60% inhibited cell growth by 40 +/- 26% compared with that of a sequence-unrelated oligonucleotide. Indomethacin had no effect on PAEC growth over a range of concentrations (0.3-5 microM). These data show that 5LO inhibitors impaired the proliferative response of the cultured PAECs, suggesting that this enzyme may contribute to PAEC growth under certain pathological conditions.