Furosemide has been given as a bronchodilator by nebulization for direct application to the airways in an attempt to localize its actions and minimize systemic effects. It is also given by systemic administration in the treatment of infants with chronic lung disease. Although clinical studies demonstrate variable results, furosemide relaxes airway smooth muscle in vitro, using isometric tension measurement. A disadvantage of this technique is that in this system, tissues are stretched in a two dimensional fashion. Therefore, we used a cannulated, perfused video micrometry system to determine if the way in which furosemide reaches the airway, by epithelial deposition or by systemic absorption, affects the amount of relaxation. Trachea (600-800 μ diameter) from newborn mice (2-4 d), C57BL/6J and AJ strains, were cannulated and mounted onto a video micrometry system (Living Systems, Burlington, VT). Tissues were bathed in and perfused with HEPES buffer, pH 7.4 at 37°C, bubbled with 100% O2. After an equilibration period, the airways were preconstricted with extraluminal 10 μM Acetylcholine. When stable tension was achieved, 300 μM furosemide was added either extraluminally or intraluminally. Effects of extraluminal and intraluminal furosemide were compared (% change in airway diameter relative to Acetylcholine preconstriction). Relaxation to furosemide given intraluminally (65 ± 9%), with direct epithelial exposure, was not different from relaxation of extraluminally (52 ± 11%) exposed airway (p = 0.4, n=7). Thus the route of furosemide administration did not change the magnitude of relaxation when the same concentration was present either intra- or extraluminally, indicating that furosemide mediated airway relaxation is non-epithelial dependent. Further, systemically administered furosemide may produce airway relaxation if adequate concentrations reach the airway. Preliminary data herein suggest that this cannulated, perfused system may allow for a more physiologic response of the intact airway and may allow for the differentiation of epithelially mediated responses. Funding: NIH-HL45220, Leahi Trust, & US Army HSC.