Several transport systems mediating the placental transport of Na, K and Cl have been described, but whether the trophoblast membrane also expresses a Na-K-Cl cotransporter that mediates the coupled movement of all three ions remains unclear. Here we show that BeWo cells, a human trophoblastic cell line, exhibit bumetanide-sensitive Rb-86 (a K surrogate) uptake. Entry via this route accounts for similar to 17% of the Rb-86 influx with the remainder being mediated largely via the Na,K-ATPase. The activity of the bumetanide-sensitive transporter was rapidly elevated (>40%) upon subjecting cells to an acute hyperosmotic challenge signifying a potential role in cell volume regulation. Antibodies to the Na-K-Cl cotransporter identified a single band of similar to 200 kDa on Western blots of fractionated BeWo membranes. This immunoreactivity colocalised with that of the Na,K-ATPase (a basal membrane marker), but was absent from membranes enriched with placental alkaline phosphatase tan apical membrane marker). These findings show for the first time, that a Na-K-Cl cotransporter is expressed in a human placental cell line which may be involved in regulating trophoblast cell volume.