Induction of programmed cell death has been described during infection with many different viruses. We have investigated the influence of African swine fever virus (ASFV) on apoptosis of different cell populations during in vitro and in vivo infection. We observed apoptosis in ASFV-infected monocyte/macrophage and peripheral blood mononuclear cell cultures. Apoptosis was demonstrated in these cells by DNA fragmentation, DNA staining and DNA-associated histone fraction detection assays. Flow cytometry analysis of infected cultures also showed morphological and functional alterations, including changes in the cell cycle and percentage of cell fractions stained with propidium iodide. After in vivo infection with three different virulent strains of ASFV, apoptosis of infected cells from the mononuclear phagocytic system and closely related elements from different tissues was observed. Additionally, infected pigs showed an intense degree of apoptosis of lymphocytes, which are not infected by the virus. In lymph nodes and other lymphoid organs, broad bands of apoptotic cells presented typical nuclear changes under light microscopy. The occurrence of DNA fragmentation was confirmed in these tissues using terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling. These findings, together with the pathological observations in infected pigs of a depletion in cell populations in lymphoid organs, suggest that virus interference with programmed cell death plays a central role in pathogenesis of this disease, being responsible for lymphoid organ impairment in acute ASFV infection.