Vascular endothelial cells have been shown previously to possess a highly active Na-K-Cl cotransport system that mediates the major portion of total K influx and is regulated by a variety of vasoactive hormones and neurotransmitters. These observations suggest that the cotransporter may be an important component of endothelial cell function. The present study was conducted to investigate the role of Na-K-Cl cotransport in regulation of endothelial cell volume. Cultured bovine aortic endothelial cells were exposed to media of varying tonicities and Na-K-Cl cotransport activity assessed as bumetanide-sensitive K influx. Increasing the extracellular tonicity by increments as small as 10 mosM was found to cause significant stimulation of cotransport activity, and lowering tonicity reduced activity of the transporter. Exposure of endothelial cells to hypertonic medium was also found to increase bumetanide-sensitive net uptake of Na and K and total cellular Na and K content. Endothelial cell volume was evaluated by [14C]urea determination of intracellular water space in endothelial monolayers and by electronic cell sizing of suspended cells. Treatment of the cells with agents that stimulate Na-K-Cl cotransport activity was found to increase cell volume, whereas cotransport-inhibiting agents decreased cell volume. Exposure of the cells to hypertonic medium caused a rapid decrease in cell volume, followed by a regulatory volume increase that was greatly attenuated by bumetanide. The volume recovery was partially inhibited by the Na-H exchange inhibitor amiloride and was nearly abolished by bumetanide and amiloride in combination. Endothelial cells of pulmonary artery and cerebral microvessels were also found to exhibit increased Na-K-Cl cotransport activity on exposure to hypertonic media. These findings suggest that Na-K-Cl cotransport is of major importance in endothelial cell volume regulation.