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 [³H]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 IC₅₀ values of 3.9, 1.8, and 0.48 µM, 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 IC₉₀ 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 G₀/G₁ phase. Antisense oligonucleotides to 5LO mRNA delivered at a transfection efficiency of ~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 µM). 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.