Since most studies addressing the regulatory mechanisms of ICAM-1 expression have used cultured endothelial cells, we set out to develop an isolated mouse lung preparation to study gene and protein expression in its proper cellular context in the organ. Lungs from CD1 mice were isolated and perfused (2 ml/min, 37°C) with a re-circulating volume of RPMI 1640 solution supplemented with 3 g/100 ml albumin. Lungs maintained their isogravimetric state for 4 hr. TNF-(2,000 U/ml) was added to the perfusate for 0.5, 1, 2, or 3.5 hr to induce ICAM-1 expression or lungs received no treatment (control). After quick-freezing the lungs using liquid nitrogen at different time points, the prepared tissue homogenates were analyzed for ICAM-1 protein expression by Western blotting and NF-B activation by electrophoretic mobility shift assay (EMSA). TNF-caused a progressive increase in NF-B activity after 0.5 hr and ICAM-1 protein expression 2- to 3-fold of basal after 2 hr. Untreated lungs expressed a low and constant level of ICAM-1 between 0 and 3.5 hr. TNF-failed to induce NF-B activation and ICAM-1 expression in lungs of NADPH oxidase-deficient mice lacking p47^phox (p47^phox-/-). We disaggregated mouse lungs using collagenase, and stained the cells for ICAM-1 and VE-cadherin (used as an endothelial marker) to assess the in situ endothelial-specific expression of ICAM-1. We observed that TNF challenge resulted in increased ICAM-1 expression in endothelial cells freshly isolated from lungs. These data show the role of NADPH oxidase-derived oxidant signaling in the mechanism of NF-B activation and ICAM-1 expression in mouse lung endothelial cells. Moreover, the general method presented herein has potential value in assessing mechanisms of gene and protein expression in the isolated-perfused mouse lung model.