One hundred fifteen samples of wolf (Canis lupus) feces were collected during 1994 to 1995 from four free-living populations of the north central Apennines Mountains, Italy. The samples were tested for canine parvovirus by antigen-capture enzyme-linked immunosorbent assay (ELISA), hemagglutination, and virus isolation. Four of these samples were positive by virus isolation as confirmed by electron microscopy. All positive samples were from Casentino Park in Tuscany. This is the first definitive observation of canine parvovirus in wolves from Europe.