Two cases of fatal infection caused by parvovirus in a white tiger (Panthera tigris) and an African lion (Panthera leo) at the Lisbon Zoo (Portugal) are described. Gross findings at necropsy were catharral enteritis in the tiger and severe hemorrhagic enteritis in the lion. Histopathologic examination revealed, in both animals, intestinal crypt necrosis and lymphocyte depletion in the germinal centers of the mesenteric lymph nodes. Bacteriologic examination was negative for common bacterial pathogens, including Salmonella. Amplification of the parvovirus VP2 complete gene was achieved in both cases and sequencing analysis identified these isolates as feline panleukopenia virus (FPLV). The nucleotide sequences obtained from these two viruses were genetically indistinguishable. The phylogenetic analysis of FPLV strains from domestic cats obtained in the Lisbon area revealed the circulation of FPLV strains highly similar to those isolated from the tiger and lion, which strongly suggests that stray cats may have been the source of infection.