Exposure of bovine pulmonary artery endothelial cells to 3% O2 resulted in approximately twofold stimulation of serotonin (5-HT) uptake after 24-48 h when compared with cells exposed to 20% O2. The enhanced uptake was reversed after 48 h when cells were again placed in 20% O2. The stimulation was not observed after 0.5 or 2 h of exposure to hypoxia. The stimulation was present when iproniazid blocked conversion of 5-HT to 5-hydroxyindole-3-acetic acid, indicating that enhanced uptake did not occur through augmentation of monoamine oxidase activity. Stimulation of uptake by hypoxia occurred at low concentrations of 5-HT (up to 10(-6) M) but not at high 5-HT concentrations (greater than 10(-5) M) and was blocked by imipramine or absence of sodium from the medium, indicating that high-affinity transport and not diffusion of 5-HT was stimulated. Furthermore, exposure of cells to hypoxia did not produce morphological evidence of injury or change in protein content or trypan blue exclusion. The cell number of 3% O2-exposed cells was slightly reduced when compared with controls after 48 h. There was no change in cellular ATP or increase in lactate dehydrogenase in medium of cells exposed to 3% O2. Thus exposure of endothelial cells in culture to hypoxia stimulates the membrane activity of 5-HT accumulation with no evidence of injury to the cell.