From January 2004 to July 2007, 2455 sera were collected from domestic and wild animals living in central Italy and tested by indirect immunofluorescence assay to detect antibodies against Anaplasma phagocytophilum. Considering sera with 1:40 antibody titers as positive, 336 (13.68%) animals scored positive. The percentages of seropositivity were: 46.26% (31/67) in fallow deer, 46.15% (24/52) in red deer, 16.89% (134/793) in horses, 16.78% (23/137) in cattle, 12.74% (13/102) in sheep, 8.76% (108/1232) in dogs, 4.16% (3/72) in goats. These data confirm the presence of A. phagocytophilum in wild ruminants and domestic animals, including pets, in central Italy.