Nitric oxide (NO) produced by endothelial cells diffuses to vascular smooth muscle cells to cause dilatation of the renal vasculature and other vessels. Although it is generally assumed that NO moves from cell to cell by free diffusion, we have recently shown that aquaporin-1 (AQP-1) transports NO across cell membranes. AQP-1 is expressed in endothelial and vascular smooth muscle cells. We hypothesized that diffusion of NO into vascular smooth muscle cells and out of endothelial cells is facilitated by AQP-1, and transport of NO by AQP-1 is involved in endothelium-dependent relaxation. In intact-aortic rings from AQP-1 -/-mice, vasorelaxation induced by acetylcholine (which increases endogenous NO) was reduced (p<0.0001 vs. control). No differences were found in the relaxation caused by intracellular delivery of NO or intracellular cGMP between strains. In endothelium-denuded aortic rings from AQP-1-/-mice, the vasorelaxant capability of NO released in the extracellular space was reduced (p<0.0001 vs. control). Influx of NO (5µM) into vascular smooth muscle cells was 0.17±0.02 f.u./sec for control and 0.07±0.01 f.u./sec for AQP-1-/- mice, 62% lower (p<0.002). NO released by endothelial cells in response to 1µM acetylcholine was 96.2±17.7 pmol NO/mg for control and 41.9±13.4 pmol NO/mg for AQP-1-/- mice, 56% reduction (p<0.04). NOS3 expression was 1.33±0.29 O.D.units for control and 3.84±0.76 O.D.units for AQP-1-/- mice, 188% increase (p<0.01). We conclude that: 1) AQP-1 facilitates NO influx into vascular smooth muscle cells; 2) AQP-1 facilitates NO diffusion out of endothelial cells; 3) transport of NO by AQP-1 is required for full expression of endothelium-dependent relaxation.