BACKGROUND: While ex vivo lung perfusion (EVLP) has become established in lung transplantation, the cellular processes occurring during this period are not yet fully understood. Prior studies demonstrated that donor leukocytes (DLs) migrate from the graft into the perfusate during EVLP, but the distribution of DLs in graft and perfusate compartments has not been characterized. Moreover, cell death of DLs has been implicated in mediating graft injury during EVLP, but the underlying mechanisms have not been elucidated. We hypothesized the following: (1) there is a nonspecific migration of DLs from the graft into perfusate and (2) cell death of DLs releases damage-associated molecular patterns (DAMPs) that contribute to the inflammatory milieu during EVLP. METHODS: EVLP was performed on rat lungs for 3 hours (N = 6). At the end of EVLP, flow cytometry was used to quantify the distribution of different DL cell types in both the graft and perfusate compartments. During EVLP, the perfusate was also sampled hourly to measure levels of DAMPs and downstream inflammatory cytokines generated during EVLP. RESULTS: At the conclusion of EVLP, there was a significantly higher proportion of T and B cells present in the perfusate compartment compared with the graft compartment. There was a time-dependent increase in extracellular DNA and tumor necrosis factor α in the perfusate during EVLP. CONCLUSIONS: T cells and B cells are enriched in the perfusate compartment during EVLP. Cell death of DLs contributes to an accumulation of DAMPs during EVLP.