Retrograde lung vascular perfusion can appear in high‐risk surgeries. The present report is the first to study long‐term retrograde perfusion of isolated perfused mouse lungs (IPLs) and to use the tyrosine kinase ephB4 and its ligand ephrinB2 as potential markers for acute lung injury. Mouse lungs were subjected to anterograde or retrograde perfusion with normal‐pressure ventilation (NV) or high‐pressure ventilation (=overventilation, OV) for 4 hours. Outcome parameters were cytokine, ephrinB2 and ephB4 levels in perfusate samples and bronchoalveolar lavage (BAL), and the wet‐to‐dry ratio. Anterograde perfusion was feasible for 4 hours, while lungs receiving retrograde perfusion presented considerable collapse rates. Retrograde perfusion resulted in an increased wet‐to‐dry ratio when combined with high‐pressure ventilation; other physiological parameters were not affected. Cytokine levels in BAL and perfusate, as well as levels of soluble ephB4 in BAL were increased in OV, while soluble ephrinB2 BAL levels were increased in retrograde perfusion. BAL levels of ephrinB2 and ephB4 were also determined in vivo, including mice ventilated for 7 hours with normal‐volume ventilation (NVV) or high‐volume ventilation (HVV) with increased levels of ephB4 in HVV BAL compared to NVV. Retrograde perfusion in IPL is limited as a routine method to investigate effects due to collapse for yet unclear reasons. If successful, retrograde perfusion has an influence on pulmonary oedema formation. In BAL, ephrinB2 seems to be up‐regulated by flow reversal, while ephB4 is a marker for acute lung injury.