The widespread presence of the Na-K-2Cl (NKCC) cotransporter protein suggests that chronic administration of inhibitors may result in adverse effects. Inhibition of the NKCC cotransporter by loop diuretics is felt to underlie the diuretic and the pulmonary smooth muscle relaxant effects of this class of drugs. However, the fundamental regulation of salt and water movement by this cotransporter suggests that it may also mediate cell volume changes that occur during cell cycle progression. Thus we hypothesized that NKCC cotransporter inhibition by loop diuretics would decrease cellular proliferation. Normal human bronchial smooth muscle cells (BSMC) showed a significant concentrationdependent decrease in cell counts after 7 days of exposure to both bumetanide (n=5-10) and furosemide (n=6-16), compared to controls. Proliferation was similarly inhibited in normal human lung fibroblasts (n=5-9). To determine if this was due to loss of cells, apoptosis assays were performed on BSMC. Both annexin V-propidium iodide staining (n=5-10) and single cell gel electrophoresis assays (n=4) were negative for necrosis and apoptosis in BSMC exposed to 10 µM bumetanide. Subsequent analysis of the cell cycle by flow cytometry showed that bumetanide exposed BSMC were delayed in G1 phase compared with controls (n=4-8). In summary, this is the first evidence for loop diuretic inhibition of airway smooth muscle cell proliferation. NKCC cotransporter inhibition impeded G1-S phase transition, but does not facilitate cell death. Thus although inhibition by loop diuretics relaxes airway smooth muscle, there may be a more important role for the NKCC cotransporter in the regulation of cell proliferation.