The objective of this study was to quantitatively assess changes in cell adhesion molecules (CAM) expression on the pulmonary endothelial surface during hyperoxia and to assess the functional significance of those changes on cellular trafficking and development of oxygen-induced lung injury. Mice were placed in >95% O₂ for 0-72 hours and pulmonary injury and neutrophil (PMN) sequestration were assessed. Specific pulmonary CAMs uptake was quantified using a dual radiolabeled monoclonal antibody (mAb) technique. To test the role of CAMs in PMN trafficking during hyperoxia, blocking mAbs to murine P-selectin, ICAM-1, or PECAM-1 were injected in wild-type mice. Mice genetically deficient in these CAMs and PMN-depleted mice were also evaluated. Pulmonary neutrophil sequestration occurred within 8 hours of hyperoxia, although alveolar emigration occurred later (between 48-72 hours), coincident with rapid escalation of the lung injury. Hyperoxia significantly increased pulmonary uptake of radiolabeled antibodies to P-selectin, ICAM-1 and PECAM-1, reflecting increase of their level on pulmonary endothelium and possibly sequestered blood cells. Although both anti-PECAM-1 and anti-ICAM-1 antibodies suppressed PMN alveolar influx in wild-type mice, only mice genetically deficient in PECAM-1 showed PMN influx suppression. Neither CAM blockade, nor genetic deficiency, nor PMN depletion attenuated lung injury. We conclude that early pulmonary PMN retention during hyperoxia is not temporally associated with an increase in endothelial CAMs; however, subsequent PMN emigration into the alveolar space may be supported by PECAM-1 and ICAM-1. Blocking neutrophil recruitment did not prevent lung injury, supporting dissociation between PMN infiltration and lung injury during hyperoxia in mice.