Inhibition of the Na-K-2Cl (NKCC) cotransporter by loop diuretics is associated with airway relaxation, but there has been no direct evidence for the expression of this protein in airway smooth muscle. Thus, we hypothesized that a NKCC cotransporter is present and functional in airway smooth muscle cells. Monoclonal and polyclonal antibodies were used first to demonstrate the presence of a NKCC cotransporter protein in isolated human fetal trachea and normal human bronchial smooth muscle cells (BSMC) by western blotting. The cotransporter protein was then localized by immunohistochemical staining to airway smooth muscle cells in culture and in situ. The localization was confirmed by indirect immunofluorescence and laser confocal microscopy in the BSMC. Cotransporter function in BSMC was also confirmed in vitro by bumetanide mediated inhibition of rubidium uptake. Our present findings thus document the presence of a functional NKCC cotransporter in human airway smooth muscle, providing a basis for defining the role of this ion cotransporter in airway smooth muscle function.