This work describes, for the first time, the changes taking place in the antioxidative system of the leaf apoplast in response to Plum pox virus (PPV) in different Prunus species showing different susceptibilities to PPV. The presence of p-hydroxy mercury benzoic acid (pHMB)-sensitive ascorbate peroxidase (APX) (Class I APX) and pHMB-insensitive APX (Class III APX), superoxide dismutase (SOD), peroxidases (POX), NADH-POX and polyphenoloxidase (PPO) was described in the apoplast from both peach and apricot leaves. PPV infection produced different changes in the antioxidant system of the leaf apoplast from the Prunus species, depending on their susceptibility to the virus. In leaves of the very susceptible peach cultivar GF305, PPV brought about an increase in Class I APX, POX, NADH-POX and PPO activities. In the susceptible apricot cultivar Real Fino, PPV infection produced a decrease in apoplastic POX and SOD activities, whereas a strong increase in PPO was observed. However, in the resistant apricot cultivar Stark Early Orange, a rise in Class I APX as well as a strong increase in POX and SOD activities was noticed in the apoplastic compartment. Long-term PPV infection produced an oxidative stress in the apoplastic space from apricot and peach plants, as observed by the increase in H2O2 contents in this compartment. However, this increase was much higher in the PPV-susceptible plants than in the resistant apricot cultivar. Only in the PPV-susceptible apricot and peach plants was the increase in apoplastic H2O2 levels accompanied by an increase in electrolyte leakage. No changes in the electrolyte leakage were observed in the PPV-inoculated resistant apricot leaves, although a 42% increase in the apoplastic H2O2 levels was produced. Two-dimensional electrophoresis analyses revealed that the majority of the polypeptides in the apoplastic fluid had isoelectric points in the range of pI 4-6. The identification of proteins using MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) and peptide mass fingerprinting analyses showed the induction of a thaumatin-like protein as well as the decrease of mandelonitrile lyase in peach apoplast due to PPV infection. However, most of the selected polypeptides showed no homology with known proteins. This fact emphasizes that, at least in Prunus, most of the functions of the apoplastic space remain unknown. We conclude that long-term PPV infection produced an oxidative stress in the leaf apoplast, contributing to the deleterious effects produced by PPV infection in leaves of inoculated, susceptible Prunus plantsThis work has been supported by grant AGL-2002-02115 from the CICYT (Comisión Interministerial de Ciencia y Tecnología) of Spain and grant 23BIO2005-04-6444 from the Comunidad de Murcia (Spain). P.M. Periago thanks the Ministerio de Ciencia y Tecnología for her contract in the Programme “Ramón y Cajal”.Peer reviewed