To evaluate the relative contributions of three possible mechanisms that can be advanced to explain the observation that hyperoxia decreases serotonin uptake by endothelial cells, we examined the effect of high O2 tensions on Na+-K+-ATPase activity, ATP content, and plasma membrane fluidity in cultured endothelial cells. Confluent monolayers of pulmonary artery and aortic endothelial cells were exposed to 95% O2 (hyperoxia) or 20% O2 (controls) in 5% CO2 at 1 ATA for 4-42 h. Exposure to high O2 tensions had no effect on Na+-K+-ATPase activity or ATP content in pulmonary artery or aortic endothelial cells in culture. However, hyperoxia decreased the fluidity of the plasma membrane of pulmonary artery and aortic endothelial cells in culture, and the time course for the decrease in fluidity parallels that of the hyperoxic inhibition of serotonin transport. These results indicate that hyperoxia decreases fluidity in the hydrophobic core of the plasma membranes of cultured endothelial cells. Such decreases in plasma membrane fluidity may be responsible for hyperoxia-induced alterations in membrane function including decreases in transmembrane transport of amines.